Carboxylesterase nucleic acid molecules, proteins and uses thereof

ABSTRACT

The present invention relates to arthropod esterase proteins; to arthropod esterase nucleic acid molecules, including those that encode such esterase proteins; to antibodies raised against such esterase proteins; and to other compounds that inhibit arthropod esterase activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to protect animals from hematophagous arthropod infestation.

This Application is a 371 of PCT/US97/20598, filed Nov. 10, 1997, whichis a continuation-in-part of application Ser. No. 08/747,221, filed Nov.12, 1996, now issued as U.S. Pat. No. 6,063,610.

FIELD OF THE INVENTION

The present invention relates to arthropod esterase nucleic acidmolecules, proteins encoded by such nucleic acid molecules, antibodiesraised against such proteins, and inhibitors of such proteins. Thepresent invention also includes therapeutic compositions comprising suchnucleic acid molecules, proteins, antibodies, and/or other inhibitors,as well as their use to protect an animal from hematophagous arthropodinfestation.

BACKGROUND OF THE INVENTION

Hematophagous arthropod infestation of animals is a health and economicconcern because hematophagous arthropods are known to cause and/ortransmit a variety of diseases. Hematophagous arthropods directly causea variety of diseases, including allergies, and also carry a variety ofinfectious agents including, but not limited to, endoparasites (e.g.,nematodes, cestodes, trematodes and protozoa), bacteria and viruses.

In particular, the bites of hematophagous arthropods are a problem foranimals maintained as pets because the infestation becomes a source ofannoyance not only for the pet but also for the pet owner who may findhis or her home generally contaminated with insects. As such,hematophagous arthropods are a problem not only when they are on ananimal but also when they are in the general environment of the animal.

Bites from hematophagous arthropods are a particular problem becausethey not only can lead to disease transmission but also can cause ahypersensitive response in animals which is manifested as disease. Forexample, bites from fleas can cause an allergic disease called fleaallergic (or allergy) dermatitis (FAD). A hypersensitive response inanimals typically results in localized tissue inflammation and damage,causing substantial discomfort to the animal.

The medical importance of arthropod infestation has prompted thedevelopment of reagents capable of controlling arthropod infestation.Commonly encountered methods to control arthropod infestation aregenerally focused on use of insecticides. While some of these productsare efficacious, most, at best, offer protection of a very limitedduration. Furthermore, many of the methods are often not successful inreducing arthropod populations. In particular, insecticides have beenused to prevent hematophagous arthropod infestation of animals by addingsuch insecticides to shampoos, powders, collars, sprays, foggers andliquid bath treatments (i.e., dips). Reduction of hematophagousarthropod infestation on the pet has been unsuccessful for one or moreof the following reasons: (1) failure of owner compliance (frequentadministration is required); (2) behavioral or physiological intoleranceof the pet to the pesticide product or means of administration; and (3)the emergence of hematophagous arthropod populations resistant to theprescribed dose of pesticide. However, hematophagous arthropodpopulations have been found to become resistant to insecticides.

Prior investigators have described insect carboxylesterase (CE) proteinbiochemistry, for example, Chen et al., Insect Biochem. Molec. Biol.,24:347-355, 1994; Whyard et al., Biochemical Genetics, 32:924, 1994 andArgentine et al., Insect Biochem. Molec Biol, 25:621-630, 1995. Otherinvestigators have disclosed certain insect CE amino acid sequences, forexample, Mouches et al., Proc Natl Acacd Sci USA, 87:2574-2578, 1990 andCooke et al., Proc Natl Acad Sci USA, 86:1426-1430, 1989, and nucleicacid sequence (Vaughn et al., J. Biol. Chem., 270:17044-17049, 1995).

Prior investigators have described certain insect juvenile hormoneesterase (JHE) nucleic acid and amino acid sequences: for example,sequence for Heliothis virescens is disclosed by Hanzlik et al., J.Biol. Chem., 264:12419-12425, 1989; Eldridge et al., App EnvironMicrobiol, 58:1583-1591, 1992; Bonning et al., Insect Biochem. Molec.Biol., 22:453-458, 1992; Bonning et al., Natural and Engineered PestManagement Agents, pp. 368-383, 1994 and Harshman et al., InsectBiochem. Molec. Biol, 24:671-676, 1994; sequence for Manduca sexta'sdisclosed by Vankatesh et al., J Biol Chem, 265:21727-21732, 1990;sequence for Trichoplusia ni is disclosed by Venkataraman et al., Dev.Genet., 15:391-400, 1994 and Jones et al., Biochem. J., 302:827-835,1994; and sequence for Lymantria dispar is disclosed by Valaitis, InsectBiochem. Molec. Biol., 22:639-648, 1992.

Identification of an esterase of the present invention is unexpected,however, because even the most similar nucleic acid sequence identifiedby previous investigators could not be used to identify an esterase ofthe present invention. In addition, identification of an esteraseprotein of the present invention is unexpected because a proteinfraction from flea prepupal larvae that was obtained by monitoring forserine protease activity surprisingly also contained esterase proteinsof the present invention.

In summary, there remains a need to develop a reagent and a method toprotect animals or plants from hematophagous arthropod infestation.

SUMMARY OF THE INVENTION

The present invention relates to a novel product and process forprotection of animals or plants from arthropod infestation. According tothe present invention there are provided arthropod esterase proteins andmimetopes thereof; arthropod nucleic acid molecules, including thosethat encode such proteins; antibodies raised against such esteraseproteins (i.e., anti-arthropod esterase antibodies); and compounds thatinhibit arthropod esterase activity (i.e, inhibitory compounds orinhibitors).

The present invention also includes methods to obtain such proteins,mimetopes, nucleic acid molecules, antibodies and inhibitory compounds.Also included in the present invention are therapeutic compositionscomprising such proteins, mimetopes, nucleic acid molecules, antibodies,and/or inhibitory compounds, as well as use of such therapeuticcompositions to protect animals from arthropod infestation.

Identification of an esterase of the present invention is unexpected,however, because the most similar nucleic acid sequence identified byprevious investigators could not be used to identify an esterase of thepresent invention. In addition, identification of an esterase protein ofthe present invention is unexpected because a protein fraction from fleaprepupal larvae that was obtained by monitoring for serine proteaseactivity surprisingly also contained esterase proteins of the presentinvention.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with agene comprising a nucleic acid sequence including SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleicacid molecule encoding a protein comprising amino acid sequence SEQ IDNO:74.

The present invention also includes a nucleic acid molecule thathybridizes under stringent hybridization conditions with a nucleic acidmolecule encoding a protein comprising at least one of the followingamino acid sequences: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74; and particularlya nucleic acid molecule that hybridizes with a nucleic acid sequencethat is a complement of a nucleic acid sequence encoding any of theamino acid sequences. A preferred nucleic acid molecule of the presentinvention includes a nucleic acid molecule comprising a nucleic acidsequence including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a proteincomprising amino acid sequence SEQ ID NO:74, and allelic variantsthereof.

The present invention also includes an isolated carboxylesterase nucleicacid molecule comprising a nucleic acid sequence encoding a proteincomprising an amino acid sequence including SEQ ID NO:5, SEQ ID NO:19,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:44 and/or SEQ ID NO:53, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 represent N-terminalamino acid sequences of carboxylesterases isolated from prepupal flealarvae, the production of which are described in the Examples of thepresent application.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include a nucleic acid molecule ofthe present invention. Also included are methods to produce such nucleicacid molecules, recombinant molecules, recombinant viruses andrecombinant cells.

Another embodiment of the present invention includes an isolatedesterase protein that is encoded by a nucleic acid molecule thathybridizes under stringent hybridization conditions to (a) a nucleicacid molecule that includes at least one of the following nucleic acidsequences: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ IDNO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:69, and SEQ ID NO:71; and/or (b) anucleic acid molecule encoding a protein including at least one of thefollowing amino acid sequences: SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55 and SEQ ID NO:74. One embodiment is acarboxylesterase protein encoded by a nucleic acid molecule thathybridizes under stringent hybridization conditions to a nucleic acidmolecule that encodes a protein comprising at least one of the followingamino acid sequences: SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQID NO:53. Preferred proteins of the present invention are isolated fleaproteins including at least one of the following amino acid sequences:SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ IDNO:73 and SEQ ID NO:74; also included are proteins encoded by allelicvariants of nucleic acid molecules encoding proteins comprising any ofthe above-listed amino acid sequences.

The present invention also relates to mimetopes of arthropod esteraseproteins as well as to isolated antibodies that selectively bind toarthropod esterase proteins or mimetopes thereof. Also included aremethods, including recombinant methods, to produce proteins, mimetopesand antibodies of the present invention.

The present invention also includes a formulation of fleacarboxylesterase proteins, in which the proteins, when submitted to 14%Tris-glycine SDS-PAGE, comprise a fractionation profile as depicted inFIG. 3, in which the proteins have carboxylesterase activity.

Also included in the present invention is a formulation of fleacarboxylesterase proteins, in which the proteins, when submitted toIEF-PAGE, comprise a fractionation profile as depicted in FIG. 4, lane3, lane 4, lane 5, lane 6 and/or lane 7, wherein the proteins havecarboxylesterase activity.

Another embodiment of the present invention is an isolated flea proteinor a formulation of flea proteins that hydrolyzes α-napthyl acetate toproduce α-napthol, when the protein is incubated in the presence ofα-napthyl acetate contained in 20 mM Tris at pH 8.0 for about 15 minutesat about 37° C.

Yet another embodiment of the present invention is an isolated fleaprotein or a formulation of flea proteins that hydrolyzes the methylester group of juvenile hormone to produce a juvenile hormone acid.

Another embodiment of the present invention is a method to identify acompound capable of inhibiting flea carboxylesterase activity, themethod comprising: (a) contacting an isolated flea carboxylesterase witha putative inhibitory compound under conditions in which, in the absenceof the compound, the protein has carboxylesterase activity; and (b)determining if the putative inhibitory compound inhibits the activity.Also included in the present invention is a test kit to identify acompound capable of inhibiting flea carboxylesterase activity, the testkit comprising an isolated flea carboxylesterase protein having esteraseactivity and a means for determining the extent of inhibition of theactivity in the presence of a putative inhibitory compound.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of reducing hematophagous ectoparasiteinfestation. Such a therapeutic composition includes at least one of thefollowing protective compounds: an isolated hematophagous ectoparasitecarboxylesterase protein or a mimetope thereof, an isolatedcarboxylesterase nucleic acid molecule that hybridizes under stringenthybridization conditions with a Ctenocephalides felis carboxylesterasegene, an isolated antibody that selectively binds to a hematophagousectoparasite carboxylesterase protein, and an inhibitor ofcarboxylesterase activity identified by its ability to inhibit theactivity of a flea carboxylesterase. A therapeutic composition of thepresent invention can also include an excipient, an adjuvant and/or acarrier. Preferred esterase nucleic acid molecule compounds of thepresent invention include naked nucleic acid vaccines, recombinant virusvaccines and recombinant cell vaccines. Also included in the presentinvention is a method to protect an animal from hematophagousectoparasite infestation comprising the step of administering to theanimal a therapeutic composition of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts SDS-PAGE analysis of DFP-labeled esterase proteins.

FIG. 2 depicts carboxylesterase activity of certain esterase proteins ofthe present invention.

FIG. 3 depicts SDS-PAGE analysis of carboxylesterase activity of certainesterase proteins of the present invention.

FIG. 4 depicts IEF analysis of certain esterase proteins of the presentinvention.

FIG. 5 depicts juvenile hormone esterase activity of certain esteraseproteins of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated arthropod esterase proteins,isolated arthropod esterase nucleic acid molecules, antibodies directedagainst arthropod esterase proteins and other inhibitors of arthropodesterase activity. As used herein, the terms isolated arthropod esteraseproteins and isolated arthropod esterase nucleic acid molecules refersto esterase proteins and esterase nucleic acid molecules derived fromarthropods and, as such, can be obtained from their natural source orcan be produced using, for example, recombinant nucleic acid technologyor chemical synthesis. Also included in the present invention is the useof these proteins, nucleic acid molecules, antibodies and inhibitors astherapeutic compositions to protect animals from hematophagousectoparasite infestation as well as in other applications, such as thosedisclosed below.

Arthropod esterase proteins and nucleic acid molecules of the presentinvention have utility because they represent novel targets foranti-arthropod vaccines and drugs. The products and processes of thepresent invention are advantageous because they enable the inhibition ofarthropod development, metamorphosis, feeding, digestion andreproduction processes that involve esterases. While not being bound bytheory, it is believed that expression of arthropod esterase proteinsare developmentally regulated, thereby suggesting that esterase proteinsare involved in arthropod development and/or reproduction. The presentinvention is particularly advantageous because the proteins of thepresent invention were identified in larval fleas, thereby suggestingthe importance of the proteins as developmental proteins.

One embodiment of the present invention is an esterase formulation thatincludes one or more esterase proteins capable of binding todiisopropylfluorophosphate (DFP). A preferred embodiment of an esteraseformulation of the present invention comprises one or more arthropodesterase proteins that range in molecular weight from about 20kilodaltons (kD) to about 200 kD, more preferably from about 40 kD toabout 100 kD, and even more preferably from about 60 kD to about 75 kD,as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis). An even more preferred formulation includes one ormore flea esterase proteins having elution (or migration) patterns asshown in FIG. 1.

Another embodiment of the present invention is a formulation comprisingone or more hematophagous ectoparasite carboxylesterase (CE) proteins.The present invention includes the discovery that such a formulation hasgeneral CE activity. General CE activity can be identified using methodsknown to those of skill in the art and described in the Examples sectionherein. A suitable formulation of the present invention comprises one ormore flea proteins capable of hydrolyzing α-napthyl acetate to produceα-napthol when the proteins are incubated in the presence of α-napthylacetate contained in 20 mm Tris at pH 8.0 for about 15 minutes at about37° C. General CE activity can be identified following such incubationby detecting the production of from about 0.3 to about 2.5 absorbanceunits in the presence of Fast Blue when measured at 590 nm.

A preferred CE formulation of the present invention includes one or moreflea CE proteins having acidic to neutral isoelectric points, or pIvalues. An isoelectric pH, or pI, value refers to the pH value at whicha molecule has no net electric charge and fails to move in an electricfield. A preferred formulation of the present invention includes one ormore proteins having a pI value ranging from about pI 2 to about 10,more preferably from about pI 3 to about 8, and even more preferablyfrom about pI 4.7 to about 5.2, as determined by IEF-PAGE.

An esterase formulation, including a CE formulation, of the presentinvention can be prepared by a method that includes the steps of: (a)preparing an extract by isolating flea tissue, homogenizing the tissueby sonication and clarifying the extract by centrifugation at a lowspeed spin, e.g., about 18,000 rpm for about 30 minutes; (b) recoveringsoluble proteins from said centrifuged extract and applying the proteinsto a p-aminobenzamidine agarose bead column; (c) recovering unboundprotein from the column and clarifying by filtration; (d) applying theclarified protein to a gel filtration column and eluting and collectingfractions with esterase activity; (e) dilayzing the eluate against 20 mMMES buffer, pH 6.0, containing 10 mM NaCl; (f) applying the dialysate toa cation exchange chromatography column, eluting protein bound to thecolumn with a linear grader of from about 10 mM NaCl to about 1 M NaClin 20 mM MES buffer, pH 6, and collecting fractions having esteraseactivity; (g) adjusting the pH of the resulting fractions to pH 7 andapplying the fractions to an anion exchange chromatography column; (h)eluting protein bound to the column with a linear gradient of from about0 to about 1 M NaCl in 25 mM Tris buffer, pH 6.8 and collectingfractions having esterase activity, such activity elutes from the columnat about 170 mM NaCl.

Tissue can be obtained from unfed fleas or from fleas that recentlyconsumed a blood meal (i.e., blood-fed fleas). Such flea tissues arereferred to herein as, respectively, unfed flea and fed flea tissue.Preferred flea tissue from which to obtain an esterase formulation ofthe present invention includes pre-pupal larval tissue, wandering flealarvae, 3rd instar tissue, fed adult tissue and unfed adult tissue.

In a preferred embodiment, a CE formulation of the present inventioncomprises a flea protein comprising amino acid sequence SEQ ID NO:5, SEQID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44 and/or SEQ ID NO:53.

Another embodiment of the present invention is a juvenile hormoneesterase (JHE) formulation comprising one or more arthropod JHEproteins, the arthropod being of the order Hemiptera, Anoplura,Malloplaga, Diptera, Siphonaptera, Parasitiformes, Acariformes andAcarina. The present invention includes the discovery that such aformulation has JHE activity. JHE activity can be identified usingmethods known to those of skill in the art and described in the Examplessection herein. A suitable formulation of the present inventioncomprises one or more arthropod proteins capable of hydrolyzing a methylester group of juvenile hormone to produce a juvenile hormone acid.Preferably, such a protein is capable of releasing of at least about 120counts per minute when such a protein is incubated in the presence of³H-juvenile hormone to create a reaction mixture, the reaction mixtureis combined with isooctane, the aqueous phase is recovered and theamount of ³H-juvenile hormone present in that phase is determined. Sucha protein is also preferably capable of causing release of methane thiolwhen such protein is incubated in the presence of methyl1-heptylthioacetothioate (HEPTAT) using the method generally disclosedin McCutohen et al., Insect Biochem. Molec. Biol., Vol. 25, No. 1, pg119-126, 1995, which is incorporated in its entirety by this reference.

In one embodiment, a juvenile hormone esterase formulation of thepresent invention comprises a protein comprising amino acid sequence SEQID NO:74.

According to the present invention, an arthropod that is not of theorder lepidoptera includes an arthropod of the order Hemiptera,Anoplura, Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformesand Acarina. Preferred arthropods include Hemiptera cimicidae, Hemipterareduviidae, Anoplura pediculidae, Anoplura pthiridae, Diptera culicidae,Diptera simuliidae, Diptera psychodidae, Diptera ceratopogonidae,Diptera chaoboridae, Diptera tabanidae, Diptera rhagionidae,athericidae, Diptera chloropidae, Diptera muscidae, Dipterahippoboscidae, Diptera calliphoridae, Diptera sarcophagidae, Dipteraoestridae, Diptera gastrophilidae, Diptera cuterebridae, Siphonapteraceratophyllidae, Siphonaptera leptopsyllidae, Siphonaptera pulicidae,Siphonaptera tungidae, Parasitiformes dermanyssidae, Acariformestetranychidae, Acariformes cheyletide, Acarifomies demodicidae,Acariformes erythracidae, Acariformes trombiculidae, Acariformespsoroptidae, Acariformes sarcoptidae, Acarina argasidae and Acarinaixodidae. Preferred Diptera muscidae include Musca, Hydrotaea, StomoxysHaematobia. Preferred Siphonaptera include Ceratophyllidae nosopsyllus,Ceratophyllidae diamanus, Ceratophyllidae ceratophyllus, Leptopsyllidaeleptopsylla, Pulicidae pulex, Pulicidae ctenocephalides, Pulicidaexenopsylla, Pulicidae echidnophaga and Tungidae tunga. PreferredParasitiformes dermanyssidae include Ornithonyssus and Liponyssoicles.Preferred Acarina include Argasidae argas, Argasidae ornithodoros,Argasidae otobius, Argasidae ixodes, Ixodidae hyalomma, Ixodidaenosomma, Ixodidae rhipicephalus, Ixodidae boophilus, Ixodidaedermacentor, Ixodidae haemaphysalus, Ixodidae amblyomma and Ixodidaeanocentor.

One embodiment of a JHE formulation of the present invention is one ormore arthropod JHE proteins that range in molecular weight from about 20kD to about 200 kD, more preferably from about 40 kD to about 100 kD,and even more preferably from about 60 kD to about 75 kD, as determinedby SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

A JHE formulator of the present invention can be prepared by a methodthat includes the steps of: (a) preparing soluble proteins fromarthropod extracts as described above for CE purification and purifyingsuch soluble proteins by gel filtration; (b) collecting fractions havingJHE activity from the gel filtration step, loading the fractions onto acation exchange column, eluting the cation exchange column with a lineargradient of from about 10 mM NaCl to about 1 M NaCl in 20 mM MES buffer,pH 6 and collecting fractions having JHE activity; (c) adjusting the pHof the collected fractions to about pH 7 are dialyzed against about 10mM phosphate buffer (pH 7.2) containing about 10 mM NaCl; (d) applyingthe dialysate to a hydroxyapatite column, eluting protein bound to thecolumn with a linear gradient of from about 10 mM phosphate buffer (pH7.2) containing 10 mM NaCl to about 0.5 M phosphate buffer (pH 6.5)containing 10 mM NaCl and collecting fractions having JHE activity; (e)dialyzing the fractions against 20 mM Tris buffer (pH 8.0) containing 10mM NaCl; (f) applying the dialysate an anion exchange chromatographycolumn and eluting protein bound to the column with a linear gradient offrom about 10 mM to about 1 M NaCl in 20 mM Tris buffer, pH 8 andcollecting fractions containing JHE activity.

A JHE formulation of the present invention can be prepared by a methodthat includes the steps of (a) preparing flea extracts as describedherein in the Examples section and applying the extract top-aminobenzamidine linked agarose beads and collecting protein not boundto the beads; (b) applying the unbound protein to a Superdex 200 HR gelfiltration column and collecting fractions having JHE activity; (c)applying the fractions to an anion exchange chromatography column,eluting the anion exchange column with a linear gradient of 0 to 1 MNaCl in 25 mM Tris buffer, pH 6.8 and collecting fractions having JHEactivity; (d) dialyzing the fractions overnight against about 1 L of 20mM Tris buffer, pH 8.0, containing 10 mM NaCl; (e) applying thedialysate to a Poros 10 HQ anion exchange column, eluting the columnwith buffer containing about 120 mM NaCl and collecting fractions havingJHE activity.

Suitable arthropods from which to isolate a JHE formulation of thepresent invention include, but are not limited to agricultural pests,stored product pests, forest pests, structural pests or animal healthpests. Suitable agricultural pests of the present invention include, butare not limited to Colorado potato beetles, corn earworms, fleahoppers,weevils, pink boll worms, cotton aphids, beet amryworms, lygus bugs,hessian flies, sod webworms, whites grubs, diamond back moths, whiteflies, plauthoppers, leafloppers, mealy bugs, mormon crickets and molecrickets. Suitable stored product pests of the present inventioninclude, but are not limited to dermestids, anobeids, saw toothed grainbeetles, indian mealmoths, flour beetles, long-horn wood boring beetlesand metallic wood boring beetles. Suitable forest pests of the presentinvention include, but are not limited to southern pine bark bettles,gypsy moths, elm beetles, ambrosia bettles, bag worms, tent worms andtussock moths. Suitable structural pests of the present inventioninclude, but are not limited to, bess beetles, termites, fire ants,carpenter ants, wasps, hornets, cockroaches, silverfish, Musca domesticaand Musca autumnalis. Suitable animal health pests of the presentinvention include, but are not limited to fleas, ticks, mosquitoes,black flies, lice, true bugs, sand flies, Psychodidae, tsetse flies,sheep blow flies, cattle grub, mites, horn flies, heel flies, deerflies, Culicoides and warble flies. Preferred arthropods from which toisolate a JHE formulation of the present invention include fleas,midges, mosquitos, sand flies, black flies, horse flies, snipe flies,louse flies, horn flies, deer flies, tsetse flies, buffalo flies, blowflies, stable flies, myiasis-causing flies, biting gnats, lice, mites,bee, wasps, ants, true bugs and ticks, preferably fleas, ticks and blowflies, and more preferably fleas. Preferred fleas from which to isolateJHE proteins include Ctenocephalides, Ceratophyllus, Diamanus,Echidnophga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus andXenopsylla. More preferred fleas include Ctenocephalides felis,Ctenocephalides canis, Ctenocephalides pulicidae, Pulex irritans,Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeushowardi, Xenopsylla cheopis and Pulex simulans, with C. felis being evenmore preferred.

Suitable tissue from which to isolate a JHE formulation of the presentinvention includes unfed fleas or fleas that recently consumed a bloodmeal (i.e., blood-fed fleas). Such flea tissues are referred to hereinas, respectively, unfed flea and fed flea tissue. Preferred flea tissuefrom which to obtain a JHE formulation of the present invention includespre-pupal larval tissue, 3rd instar tissue, fed or unfed adult tissue,with unfed adult gut tissue being more preferred than fed or unfed wholeadult tissue. It is of note that a JHE formulation of the presentinvention obtained from pre-pupal larval tissue does not hydrolyzeα-napthyl acetate.

Another embodiment of the present invention is an esterase formulationcomprising a combination of one or more arthropod CE and JHE proteins ofthe present invention. Suitable arthropods from which to isolate acombined CE and JHE formulation include those arthropods describedherein for the isolation of a JHE formulation of the present invention.Preferred arthropods from which to isolate a combined CE and JHEformulation include fleas, midges, mosquitos, sand flies, black flies,horse flies, horn flies, deer flies, tsetse flies, buffalo flies, blowflies, stable flies, mylasis-causing flies, biting gnats, lice, bee,wasps, ants, true bugs and ticks, preferably fleas, ticks and blowflies, and more preferably fleas. Suitable flea tissue from which toisolate a combined CE and JHE formulation of the present inventionincludes 3rd instar tissue, fed or unfed adult tissue and unfed adulttissue, with unfed adult gut tissue being more preferred than fed orunfed whole adult tissue.

In one embodiment, a formulation of the present invention comprises anesterase having both CE and JHE activity. Preferably, a formulation ofthe present invention that comprises an esterase having both CE and JHEactivity comprises a flea protein comprising amino acid sequence SEQ IDNO:8 and/or SEQ ID NO:37.

Another embodiment of the present invention is an isolated proteincomprising an arthropod esterase protein. It is to be noted that theterm “a” or “an” entity refers to one or more of that entity; forexample, a protein refers to one or more proteins or at least oneprotein. As such, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, and “having” can be usedinterchangeably. Furthermore, a compound “selected from the groupconsisting of” refers to one or more of the compounds in the list thatfollows, including mixtures (i.e., combinations) of two or more of thecompounds. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. As such, “isolated” and “biologically pure” do notnecessarily reflect the extent to which the protein has been purified.An isolated protein of the present invention can be obtained from itsnatural source, can be produced using recombinant DNA technology or canbe produced by chemical synthesis.

As used herein, an isolated arthropod esterase protein can be afull-length protein or any homolog of such a protein. An isolatedprotein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against arthropod esterase proteins, to hydrolyzeα-napthyl acetate, to hydrolyze the methyl ester group of juvenilehormone or bind to DFP. Esterase proteins of the present inventioninclude CE and JHE proteins. As such, an esterase protein of the presentinvention can comprise a protein capable of hydrolyzing α-napthylacetate, hydrolyzing the methyl ester group of juvenile hormone and/orbinding to DFP. Examples of esterase homologs include esterase proteinsin which amino acids have been deleted (e.g., a truncated version of theprotein, such as a peptide), inserted, inverted, substituted and/orderivatized (e.g., by glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitoylation, amidation and/or additionof glycerophosphatidyl inositol) such that the homolog includes at leastone epitope capable of eliciting an immune response against an arthropodesterase protein. That is, when the homolog is administered to an animalas an immunogen, using techniques known to those skilled in the art, theanimal will produce an immune response against at least one epitope of anatural arthropod esterase protein. The ability of a protein to effectan immune response, can be measured using techniques known to thoseskilled in the art. Esterase protein homologs of the present inventionalso include esterase proteins that hydrolyze α-napthyl acetate and/orthat hydrolyze the methyl ester group of juvenile hormone.

Arthropod esterase protein homologs can be the result of natural allelicvariation or natural mutation. Esterase protein homologs of the presentinvention can also be produced using techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant nucleic acid techniques to effect random ortargeted mutagenesis.

Isolated esterase proteins of the present invention have the furthercharacteristic of being encoded by nucleic acid molecules that hybridizeunder stringent hybridization conditions to a gene encoding aCtenocephalides felis protein (i.e., a C. felis esterase gene). As usedherein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules, includingoligonucleotides, are used to identify similar nucleic acid molecules.Such standard conditions are disclosed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press,1989; Sambrook et al., ibid., is incorporated by reference herein in itsentirety. Stringent hybridization conditions typically permit isolationof nucleic acid molecules having at least about 70% nucleic acidsequence identity with the nucleic acid molecule being used to probe inthe hybridization reaction. Formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permitting30% or less mismatch of nucleotides are disclosed, for example, inMeinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinikoth et al.,ibid., is incorporated by reference herein in its entirety.

As used herein, a C. felis esterase gene includes all nucleic acidsequences related to a natural C. felis esterase gene such as regulatoryregions that control production of the C. felis esterase protein encodedby that gene (such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself.In one embodiment, a C. felis esterase gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid moleculeencoding a protein comprising amino acid sequence SEQ ID NO:74. Nucleicacid sequence SEQ ID NO:1 represents the deduced sequence of the codingstrand of a PCR amplified nucleic acid molecule denoted herein asnfE1₄₀₁, the production of which is disclosed in the Examples. Thecomplement of SEQ ID NO:1 (represented herein by SEQ ID NO:3) refers tothe nucleic acid sequence of the strand complementary to the strandhaving SEQ ID NO:1, which can easily be determined by those skilled inthe art. Likewise, a nucleic acid sequence complement of any nucleicacid sequence of the present invention refers to the nucleic acidsequence of the nucleic acid strand that is complementary to (i.e., canform a complete double helix with) the strand for which the sequence iscited.

Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of thecoding strand of a PCR amplified nucleic acid molecule denoted herein asnfE2₃₆₄, the production of which is disclosed in the Examples. Thecomplement of SEQ ID NO:4 is represented herein by SEQ ID NO:6.

Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of thecoding strand of a PCR amplified nucleic acid molecule denoted herein asnfE3₄₂₁, the production of which is disclosed in the Examples. Thecomplement of SEQ ID NO:7 is represented herein by SEQ ID NO:9.

Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence ofthe coding strand of a PCR amplified nucleic acid molecule denotedherein as nfE4₅₂₄, the production of which is disclosed in the Examples.The complement of SEQ ID NO:10 is represented herein by SEQ ID NO:12.

Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence ofthe coding strand of an apparent coding region of a complementary DNA(cDNA) nucleic acid molecule denoted herein as nfE5₁₉₈₂, the productionof which is disclosed in the Examples. The complement of SEQ ID NO:13 isrepresented herein by SEQ ID NO:15.

Nucleic acid sequence SEQ ID NO:18 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE6₁₇₉₂, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:18 is representedherein by SEQ ID NO:20.

Nucleic acid sequence SEQ ID NO:24 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE7₂₈₃₆, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:24 is representedherein by SEQ ID NO:26.

Nucleic acid sequence SEQ ID NO:30 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE8₂₈₀₁, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:30 is representedherein by SEQ ID NO:32.

Nucleic acid sequence SEQ ID NO:36 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE9₂₀₀₇, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:36 is representedherein by SEQ ID NO:38.

Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE5₂₁₄₄, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:57 is representedherein by SEQ ID NO:59.

Nucleic acid sequence SEQ ID NO:67 represents the deduced sequence ofthe coding strand of an apparent coding region of a cDNA nucleic acidmolecule denoted herein as nfE10₁₉₈₇, the production of which isdisclosed in the Examples. The complement of SEQ ID NO:67 is representedherein by SEQ ID NO:69.

It should be noted that since nucleic acid sequencing technology is notentirely error-free, the nucleic acid sequences and amino acid sequencespresented herein represent, respectively, apparent nucleic acidsequences of nucleic acid molecules of the present invention andapparent amino acid sequences of esterase proteins of the presentinvention.

In another embodiment, a C. felis esterase gene can be an allelicvariant that includes a similar but not identical sequence to SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52,SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76and/or a nucleic acid molecule encoding a protein comprising amino acidsequence SEQ ID NO:74. An allelic variant of a C. felis esterase gene isa gene that occurs at essentially the same locus (or loci) in the genomeas the gene including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid moleculeencoding a protein comprising amino acid sequence SEQ ID NO:74, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art and would beexpected to be found within a given arthropod since the genome isdiploid and/or among a group of two or more arthropods.

The minimal size of an esterase protein homolog of the present inventionis a size sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid (i.e., hybridize under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. As such, the size of thenucleic acid molecule encoding such a protein homolog is dependent onnucleic acid composition and percent homology between the nucleic acidmolecule and complementary sequence. It should also be noted that theextent of homology required to form a stable hybrid can vary dependingon whether the homologous sequences are interspersed throughout thenucleic acid molecules or are clustered (i.e., localized) in distinctregions on the nucleic acid molecules. The minimal size of such nucleicacid molecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode an esterase proteinhomolog of the present invention is from about 12 to about 18nucleotides in length. Thus, the minimal size of an esterase proteinhomolog of the present invention is from about 4 to about 6 amino acidsin length. There is no limit, other than a practical limit, on themaximal size of such a nucleic acid molecule in that the nucleic acidmolecule can include a portion of a gene, an entire gene, multiplegenes, or portions thereof. The preferred size of a protein encoded by anucleic acid molecule of the present invention depends on whether afull-length, fusion, multivalent, or functional portion of such aprotein is desired.

One embodiment of the present invention includes an arthropod esteraseprotein having CE enzyme activity. Such a CE protein preferablyincludes: a catalytic triad of serine—histidine—glutamic acid as well asthe essential amino acids arginine and aspartic acid at positionssimilar to those described for juvenile hormone esterase, for example byWard et al., 1992, Int J Biochem 24: 1933-1941; this reference isincorporated by reference herein in its entirety. Analysis of theapparent full-length protein sequences disclosed herein indicates thateach of these amino acid sequences includes these amino acid motifs, aswell as surrounding consensus sequences.

Suitable arthropods from which to isolate esterase proteins havinggeneral CE activity of the present invention (including isolation of thenatural protein or production of the protein by recombinant or synthetictechniques) preferably include insects and acarines but not Culicidae,Drosophilidae, Calliphoridae, Sphingidae, Lymantriidae, Noctuidae,Fulgoroidae and Aphididae. Preferred arthropods from which to isolate CEproteins having general CE activity include fleas, ticks, black flies,lice, true bugs, sand flies, Psychodidae, tsetse flies, cattle grub,mites, horn flies, heel flies, deer flies, Culicoides and warble flies.Preferred arthropods from which to isolate an esterase proteins havinggeneral CE activity include fleas, midges, sand flies, black flies,horse flies, snipe flies, louse flies, horn flies, deer flies, tsetseflies, buffalo flies, blow flies, stable flies, myiasis-causing flies,biting gnats, lice, mites, bee, wasps, ants, true bugs and ticks,preferably fleas, ticks and blow flies, and more preferably fleas.Preferred fleas from which to isolate esterase proteins having generalCE activity include Ctenocephalides, Ceratophyllus, Diamanus,Echidnophga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus andXenopsylla. More preferred fleas include Ctenocephalides felis,Ctenocephalides canis, Ctenocephalides pulicidae, Pulex irritans,Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeushowardi, Xenopsylla cheopis and Pulex simulans, with C. felis being evenmore preferred.

A preferred arthropod esterase protein of the present invention is acompound that when administered to an animal in an effective manner, iscapable of protecting that animal from hematophagous ectoparasiteinfestation. In accordance with the present invention, the ability of anesterase protein of the present invention to protect an animal fromhematophagous ectoparasite infestation refers to the ability of thatprotein to, for example, treat, ameliorate and/or prevent infestationcaused by hematophagous arthropods. In particular, the phrase “toprotect an animal from hematophagous ectoparasite infestation” refers toreducing the potential for hematophagous ectoparasite populationexpansion on and around the animal (i.e., reducing the hematophagousectoparasite burden). Preferably, the hematophagous ectoparasitepopulation size is decreased, optimally to an extent that the animal isno longer bothered by hematophagous ectoparasites. A host animal, asused herein, is an animal from which hematophagous ectoparasites canfeed by attaching to and feeding through the skin of the animal.Hematophagous ectoparasites, and other ectoparasites, can live on a hostanimal for an extended period of time or can attach temporarily to ananimal in order to feed. At any given time, a certain percentage of ahematophagous ectoparasite population can be on a host animal whereasthe remainder can be in the environment of the animal. Such anenvironment can include not only adult hematophagous ectoparasites, butalso hematophagous ectoparasite eggs and/or hematophagous ectoparasitelarvae. The environment can be of any size such that hematophagousectoparasites in the environment are able to jump onto and off of a hostanimal. For example, the environment of an animal can include plants,such as crops, from which hematophagous ectoparasites infest an animal.As such, it is desirable not only to reduce the hematophagousectoparasite burden on an animal per se, but also to reduce thehematophagous ectoparasite burden in the environment of the animal. Inone embodiment, an esterase protein of the present invention can elicitan immune response (including a humoral and/or cellular immune response)against a hematophagous ectoparasite.

Suitable hematophagous ectoparasites to target include any hematophagousectoparasite that is essentially incapable of infesting an animaladministered an esterase protein of the present invention. As such, ahematophagous ectoparasite to target includes any hematophagousectoparasite that produces a protein having one or more epitopes thatcan be targeted by a humoral and/or cellular immune response against anesterase protein of the present invention and/or that can be targeted bya compound that otherwise inhibits esterase activity (e.g., a compoundthat inhibits hydrolysis of α-napthyl acetate, hydrolysis of the methylester group of juvenile hormone, and/or binds to DFP), thereby resultingin the decreased ability of the hematophagous ectoparasite to infest ananimal. Preferred hematophagous ectoparasite to target includeectoparasites disclosed herein as being useful in the production ofesterase proteins of the present invention.

The present invention also includes mimetopes of esterase proteins ofthe present invention. As used herein, a mimetope of an esterase proteinof the present invention refers to any compound that is able to mimicthe activity of such a protein (e.g., ability, to elicit an immuneresponse against an arthropod esterase protein of the present inventionand/or ability to inhibit esterase activity), often because the mimetopehas a structure that mimics the esterase protein. It is to be noted,however, that the mimetope need not have a structure similar to anesterase protein as long as the mimetope functionally mimics theprotein. Mimetopes can be, but are not limited to: peptides that havebeen modified to decrease their susceptibility to degradation;anti-idiotypic and/or catalytic antibodies, or fragments thereof;non-proteinaceous immunogenic portions of an isolated protein (e.g.,carbohydrate structures); synthetic or natural organic or inorganicmolecules, including nucleic acids; and/or any other peptidomimeticcompounds. Mimetopes of the present invention can be designed usingcomputer-generated structures of esterase proteins of the presentinvention. Mimetopes can also be obtained by generating random samplesof molecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner, (e.g., an esterasesubstrate, an esterase substrate analog, or an anti-esterase antibody).A preferred mimetope is a peptidomimetic compound that is structurallyand/or functionally similar to an esterase protein of the presentinvention, particularly to the active site of the esterase protein.

The present invention also includes mimetopes of esterase proteins ofthe present invention. As used herein, a mimetope of an esterase proteinof the present invention refers to any compound that is able to mimicthe activity of such an esterase protein, often because the mimetope hasa structure that mimics the esterase protein. Mimetopes can be, but arenot limited to: peptides that have been modified to decrease theirsusceptibility to degradation; anti-idiotypic and/or catalyticantibodies, or fragments thereof; non-proteinaceous immunogenic portionsof an isolated protein (e.g., carbohydrate structures); and synthetic ornatural organic molecules, including nucleic acids. Such mimetopes canbe designed using computer-generated structures of proteins of thepresent invention. Mimetopes can also be obtained by generating randomsamples of molecules, such as oligonucleotides, peptides or otherorganic molecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

One embodiment of an arthropod esterase protein of the present inventionis a fusion protein that includes an arthropod esteraseprotein-containing domain attached to one or more fusion segments.Suitable fusion segments for use with the present invention include, butare not limited to, segments that can: enhance a protein's stability;act as an immunopotentiator to enhance an immune response against anesterase protein; and/or assist purification of an esterase protein(e.g., by affinity chromatography). A suitable fusion segment can be adomain of any size that has the desired function (e.g., impartsincreased stability, imparts increased immunogenicity to a protein,and/or simplifies purification of a protein). Fusion segments can bejoined to amino and/or carboxyl termini of the esterase-containingdomain of the protein and can be susceptible to cleavage in order toenable straight-forward recovery of an esterase protein. Fusion proteinsare preferably produced by culturing a recombinant cell transformed witha fusion nucleic acid molecule that encodes a protein including thefusion segment attached to either the carboxyl and/or amino terminal endof an esterase-containing domain. Preferred fusion segments include ametal binding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); and/or a “tag” domain (e.g., at leasta portion of β-galactosidase, a strep tag peptide, other domains thatcan be purified using compounds that bind to the domain, such asmonoclonal antibodies). More preferred fusion segments include metalbinding domains, such as a poly-histidine segment; a amaltose bindingdomain; a strep tag peptide, such as that available from Biometra inTampa, Fla.; and an S10 peptide. Examples of particularly preferredfusion proteins of the present invention include PHIS-PfE6₅₄₀,PHIS-PfE7₂₇₅, PHIS-PfE7₅₇₀, PHIS-PfE8₅₇₀ and PHIS-PfE9₅₂₈, production ofwhich are disclosed herein.

In another embodiment, an arthropod esterase protein of the presentinvention also includes at least one additional protein segment that iscapable of protecting an animal from hematophagous ectoparasiteinfestations. Such a multivalent protective protein can be produced byculturing a cell transformed with a nucleic acid molecule comprising twoor more nucleic acid domains joined together in such a manner that theresulting nucleic acid molecule is expressed as a multivalent protectivecompound containing at least two protective compounds, or portionsthereof, capable of protecting an animal from hematophagous ectoparasiteinfestation by, for example, targeting two different arthropod proteins.

Examples of multivalent protective compounds include, but are notlimited to, an esterase protein of the present invention attached to oneor more compounds protective against one or more arthropod compounds.Preferred second compounds are proteinaceous compounds that effectactive immunization (e.g., antigen vaccines), passive immunization(e.g., antibodies), or that otherwise inhibit a arthropod activity thatwhen inhibited can reduce hematophagous ectoparasite burden oil andaround an animal. Examples of second compounds include a compound thatinhibits binding between an arthropod protein and its ligand (e.g., acompound that inhibits flea ATPase activity or a compound that inhibitsbinding of a peptide or steroid hormone to its receptor), a compoundthat inhibits hormone (including peptide or steroid hormone) synthesis,a compound that inhibits vitellogenesis (including production ofvitellin and/or transport and maturation thereof into a major egg yolkprotein), a compound that inhibits fat body function, a compound thatinhibits muscle action, a compound that inhibits the nervous system, acompound that inhibits the immune system and/or a compound that inhibitshematophagous ectoparasite feeding. Examples of second compounds alsoinclude proteins obtained from different stages of hematophagousectoparasite development. Particular examples of second compoundsinclude, but are not limited to, serine proteases, cysteine proteases,aminopeptidases, serine protease inhibitor proteins, calreticulins,larval serum proteins and echdysone receptors, as well as antibodies toand inhibitors of such proteins. In one embodiment, an arthropodesterase protein of the present invention is attached to one or moreadditional compounds protective against hematophagous ectoparasiteinfestation. In another embodiment, one or more protective compounds,such as those listed above, can be included in a multivalent vaccinecomprising an arthropod esterase protein of the present invention andone or more other protective molecules as separate compounds.

A preferred isolated protein of the present invention is a proteinencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with nucleic acid molecules nfE1₄₀₁, nfE2₃₆₄,nfE3₄₂₁, nfE4₅₂₄, nfE5₁₉₈₂, nfE5₁₅₁₅, nfE5₂₁₄₄, nfE5₁₆₅₀, nfE6₁₄₈₈,nfE6₁₇₉₂, nfE6₁₆₅₀, nfE7₂₈₃₆, nfE7₁₇₈₈, nfE7₁₇₁₀, nfE7₆₅₀, nfE8₂₈₀₁,nfE8₁₇₈₅, nfE8₁₇₁₀, nfE9₂₀₀₇, nfE9₁₅₈₄, nfE9₁₅₄₀, nfE10₁₉₈₇ and/ornfE10₁₅₉₀. A further preferred isolated protein is encoded by a nucleicacid molecule that hybridizes under stringent hybridization conditionswith a nucleic acid molecule having nucleic acid sequence SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ IDNO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:69 and/or SEQ ID NO:71.

Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1₄₀₁encodes a non-full-length arthropod esterase protein of about 103 aminoacids, referred to herein as PfE1₁₀₃, represented by SEQ ID NO:2,assuming the first codon spans from nucleotide 92 through nucleotide 94of SEQ ID NO:1.

Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acidsequence of PfE1₁₀₃) with amino acid sequences reported in GenBankindicates that SEQ ID NO:2, showed the most homology, i.e., about 33%identity, between SEQ ID NO:2 and alpha esterase protein from Drosophilamelanogaster.

Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2₃₆₄encodes a non-full-length arthropod esterase protein of about 121 aminoacids, referred to herein as PfE2₁₂₁, represented by SEQ ID NO:5,assuming the first codon spans from nucleotide 2 through nucleotide 4 ofSEQ ID NO:4.

Comparison of amino acid sequence SEQ ID NO:5 (i.e., the amino acidsequence of PfE2₁₂₁) with amino acid sequences reported in GenBankindicates that SEQ ID NO:5, showed the most homology, i.e., about 38%identity, between SEQ ID NO:5 and alpha esterase protein from Drosophilamelanogaster.

Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3₄₂₁encodes a non-full-length arthropod esterase protein of about 103 aminoacids, referred to herein as PfE3₁₀₃, represented by SEQ ID NO:8,assuming the first codon spans from nucleotide 113 through nucleotide115 of SEQ ID NO:7.

Comparison of amino acid sequence SEQ ID NO:8 (i.e., the amino acidsequence of PfE3₁₀₃) with amino acid sequences reported in GenBankindicates that SEQ ID NO:8, showed the most homology, i.e., about 39%identity, between SEQ ID NO:8 and alpha esterase protein from Drosophilamelanogaster.

Translation of SEQ ID NO:10 suggests that nucleic acid molecule nfE4₅₂₄encodes a non-full-length arthropod esterase protein of about 137 aminoacids, referred to herein as PfE4₁₃₇, represented by SEQ ID NO:11,assuming the first codon spans from nucleotide 113 through nucleotide115 of SEQ ID NO:10.

Comparison of amino acid sequence SEQ ID NO:11 (i.e., the amino acidsequence of PfE4₁₃₇) with amino acid sequences reported in GenBankindicates that SEQ ID NO:11, showed the most homology, i.e., about 30%identity, between SEQ ID NO:11 and Leptinotarsa decemlineataacetylcholinesterase.

Translation of SEQ ID NO:57 suggests that nucleic acid molecule nfE5₂₁₄₄encodes a full-length arthropod esterase protein of about 550 aminoacids, referred to herein as nfE8₅₅₀, represented by SEQ ID NO:58,assuming an open reading frame in which the initiation codon spans fromnucleotide 30 through nucleotide 32 of SEQ ID NO:57 and the termination(stop) codon spans from nucleotide 1680 through nucleotide 1682 of SEQID NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ IDNO:59. The coding region encoding PfE5₅₅₀ is represented by the nucleicacid molecule nfE5₁₆₅₀, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:60 and a complementary strand withnucleic acid sequence SEQ ID NO:61. The deduced amino acid sequence ofPfE5₅₅₀ (i.e., SEQ ID NO:58) predicts that PfE5₅₅₀ has an estimatedmolecular weight of about 61.8 kD and an estimated pI of about 5.5.

Comparison of amino acid sequence SEQ ID NO:58 (i.e., the amino acidsequence of PfE5₅₅₀) with amino acid sequences reported in GenBankindicates that SEQ ID NO:58 showed the most homology, i.e., about 36%identity between SEQ ID NO:58 and Drosophila melanogaster alpha esteraseprotein.

Translation of SEQ ID NO:18 suggests that nucleic acid molecule nfE6₁₇₉₂encodes a full-length arthropod esterase protein of about 550 aminoacids, referred to herein as PfE6₅₅₀, represented by SEQ ID NO:19,assuming an open reading frame having an initiation codon spanning fromnucleotide 49 through nucleotide 51 of SEQ ID NO:18 and a stop codonspanning from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18.The coding region encoding PfE6₅₅₀, is represented by nucleic acidmolecule nfE6₁₆₅₀, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:21 and a complementary strand with nucleic acidsequence SEQ ID NO:22. The proposed mature protein, denoted herein asPfE6₅₃₀, contains about 530 amino acids which is represented herein asSEQ ID NO:53. The nucleic acid molecule encoding PfE6₅₃₀ is denotedherein as nfE6₁₅₉₀ and has a coding strand having the nucleic acidsequence SEQ ID NO:23. The deduced amino acid sequence SEQ ID NO:19suggests a protein having a molecular weight of about 61.8 kD and anestimated pI of about 5.5.

Comparison of amino acid sequence SEQ ID NO:19 (i.e., the amino acidsequence of PfE6₅₅₀) with amino acid sequences reported in GenBankindicates that SEQ ID NO:19 showed the most homology, i.e., about 28%identity between SEQ ID NO:19 and Drosophila melanogaster alpha esteraseprotein.

Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE7₂₈₃₆encodes a full-length arthropod esterase protein of about 596 aminoacids, referred to herein as PfE7₅₉₆, represented by SEQ ID NO:25,assuming an open reading frame having an initiation codon spanning fromnucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codonspanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:24.The coding region encoding PfE7₅₉₆, is represented by nucleic acidmolecule nfE7₁₇₈₈, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:28 and a complementary strand with nucleic acidsequence SEQ ID NO:29. The proposed mature protein, denoted herein asPfE7₅₇₀, contains about 570 amino acids which is represented herein asSEQ ID NO:54. The nucleic acid molecule encoding PfE₇₅₇₀ is denotedherein as nfE7₁₇₁₀ and has a coding strand having the nucleic acidsequence SEQ ID NO:27. The deduced amino acid sequence SEQ ID NO:25suggests a protein having a molecular weight of about 68.7 kD and anestimated pI of about 6.1.

Comparison of amino acid sequence SEQ ID NO:25 (i.e., the amino acidsequence of PfE7596) with amino acid sequences reported in GenBankindicates that SEQ ID NO:25 showed the most homology, i.e., about 27%identity between SEQ ID NO:25 and Drosophila melanogaster alpha esteraseprotein.

Translation of SEQ ID NO:30 suggests that nucleic acid molecule nfE8₂₈₀₁encodes a full-length arthropod esterase protein of about 595 aminoacids, referred to herein as PfE8₅₉₅, represented by SEQ ID NO:31,assuming an open reading frame having an initiation codon spanning fromnucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop codonspanning from nucleotide 1884 through nucleotide 1886 of SEQ ID NO:30.The coding region encoding PfE8₅₉₅, is represented by nucleic acidmolecule nfE8₁₇₈₅, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:34 and a complementary strand with nucleic acidsequence SEQ ID NO:35. The proposed mature protein, denoted herein asPfE8₅₇₀, contains about 570 amino acids which is represented herein asSEQ ID NO:55. The nucleic acid molecule encoding PfE8₅₇₀ is denotedherein as nf8₁₇₁₀ and has a coding strand having the nucleic acidsequence SEQ ID NO:33. The deduced amino acid sequence SEQ ID NO:31suggests a protein having a molecular weight of about 68.6 kD and anestimated pI of about 6.1.

Comparison of amino acid sequence SEQ ID NO:31 (i.e., the amino acidsequence of PfE8₅₉₅) with amino acid sequences reported in GenBankindicates that SEQ ID NO:31 showed the most homology, i.e., about 28%identity between SEQ ID NO:31 and estalpha-2 esterase of Culex pipiensquinquefasciatus.

Translation of SEQ ID NO:36 suggests that nucleic acid molecule nfE9₂₀₀₇encodes a full-length arthropod esterase protein of about 528 aminoacids, referred to herein as PfE9₅₂₈, represented by SEQ ID NO:37,assuming an open reading frame having an initiation codon spanning fromnucleotide 11 through nucleotide 13 of SEQ ID NO:36 and a stop codonspanning from nucleotide 1595 through nucleotide 1597 of SEQ ID NO:36.The coding region encoding PfE9₅₂₈, is represented by nucleic acidmolecule nfE9₁₅₈₄, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:51 and a complementary strand with nucleic acidsequence SEQ ID NO:52. The deduced amino acid sequence SEQ ID NO:37suggests a protein hatting a molecular weight of about 60 kD and anestimated pI of about 5.43.

Comparison of amino acid sequence SEQ ID NO:37 (i.e., the amino acidsequence of PfE9₅₂₈) with amino acid sequences reported in GenBankindicates that SEQ ID NO:37 showed the most homology, i.e., about 37%identity between SEQ ID NO:37 and alpha esterase protein from Drosophilamelanogaster.

Translation of SEQ ID NO:67 suggests that nucleic acid moleculenfE10₁₉₈₇ encodes a full-length flea esterase protein of about 530 aminoacids, referred to herein as PfE10₅₃₀, having amino acid sequence SEQ IDNO:68, assuming an open reading frame in which the initiation codonspans from nucleotide 231 through nucleotide 233 of SEQ ID NO:67 and astop codon spanning from nucleotide 1821 through nucleotide 1823 of SEQID NO:67. The complement of SEQ ID NO:67 is represented herein by SEQ IDNO:69. The coding region encoding PfE10₅₃₀, is represented by nucleicacid molecule nfE10₁₅₉₀, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:70 and a complementary strand withnucleic acid sequence SEQ ID NO:71. The amino acid sequence of PfE10₅₃₀(i.e., SEQ ID NO:68) predicts that PfE10₅₃₀ has an estimated molecularweight of about 59.5 kD and an estimated pI of about 5.5.

Comparison of amino acid sequence SEQ ID NO:68 (i.e., the amino acidsequence of PfE10₅₃₀) with amino acid sequences reported in GenBankindicates that SEQ ID NO:68 showed the most homology, i.e., about 30%identity between SEQ ID NO:68 and Culex pipeis esterase b1 precurserprotein (swissprot #P16854).

More preferred arthropod esterase proteins of the present inventioninclude proteins comprising amino acid sequences that are at least about40%, preferably at least about 45%, more preferably at least about 50%,even more preferably at least about 55%, even more preferably at leastabout 60%, even more preferably at least about 70%, even more preferablyat least about 80%, even more preferably at least about 90%, and evenmore preferably at least about 95%, identical to amino acid sequence SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ IDNO:73 and/or SEQ ID NO:74.

More preferred arthropod esterase proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprising at leasta portion of nfE1₄₀₁, nfE2₃₆₄, nfE3₄₂₁, nfE4₅₂₄, nfE5₁₉₈₂, nfE5₁₅₁₅,nfE5₂₁₄₄, nfE5₁₆₅₀, nfE6₁₄₈₈, nfE6₁₇₉₂, nfE6₁₆₅₀, nfE7₂₈₃₆, nfE7₁₇₈₈,nfE7₁₇₁₀, nfE7₆₅₀, nfE8₂₈₀₁, nfE8₁₇₈₅, nfE8₁₇₁₀, nfE9₁₅₈₄, nfE9₁₅₄₀,nfE10₁₉₈₇ and/or nfE10₁₅₉₀, or of allelic variants of such nucleic acidmolecules. More preferred is an esterase protein encoded by nfE1₄₀₁,nfE2₃₆₄, nfE3₄₂₁, nfE4₅₂₄, nfE5₁₉₈₂, nfE5₁₅₁₅, nfE5₂₁₄₄, nfE5₆₅₀,nfE6₁₄₈₈, nfE6₁₇₉₂, nfE6₁₆₅₀, nfE7₂₈₃₆, nfE7₁₇₈₈, nfE7₁₇₁₀, nfE7₆₅₀,nfE8₂₈₀₁, nfE8₁₇₈₅, nfE8₁₇₁₀, nfE9₂₀₀₇, nfE9₁₅₈₄, nfE9₁₅₄₀, nfE10₁₉₈₇and/or nfE10₁₅₉₀, or by an allelic variant of such nucleic acidmolecules. Particularly preferred arthropod esterase proteins arePfE1₁₀₃, PfE2₁₂₁, PfE3₁₀₃, PfE4₁₃₇, PfE5₅₀₅, PfE5₅₅₀, PfE6₅₅₀, PfE6₅₃₀,PfE7₅₉₆, PfE7₅₇₀, PfE8₅₉₅, PfE8₅₇₀, PfE9₅₂₈ and PfE10₅₃₀.

In one embodiment, a preferred esterase protein of the present inventionis encoded by at least a portion of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ IDNO:57, SEQ ID NO:60 and/or SEQ ID NO:67, and, as such, has an amino acidsequence that includes at least a portion of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQID NO:31, SEQ ID NO:37, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:58 and/or SEQ ID NO:68. Also preferred is a protein encoded by anallelic variant of a nucleic acid molecule comprising at least a portionof the above-listed nucleic acid sequences.

Particularly preferred esterase proteins of the present inventioninclude SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ IDNO:68, SEQ ID NO:73 and/or SEQ ID NO:74 (including, but not limited to,the proteins consisting of such sequences, fusion proteins andmultivalent proteins) and proteins encoded by allelic variants of SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60 and/or SEQ ID NO:67.

Another embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with aC. felis esterase gene. The identifying characteristics of such a geneare heretofore described. A nucleic acid molecule of the presentinvention can include an isolated natural arthropod esterase gene or ahomolog thereof, the latter of which is described in more detail below.A nucleic acid molecule of the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a nucleic acid molecule of thepresent invention is the minimal size that can form a stable hybrid witha C. felis esterase gene under stringent hybridization conditions.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated arthropod esterase nucleic acidmolecule of the present invention can be isolated from its naturalsource or can be produced using recombinant DNA technology (e.g.,polymerase chain reaction (PCR) amplification, cloning) or chemicalsynthesis. Isolated esterase nucleic acid molecules can include, forexample, natural allelic variants and nucleic acid molecules modified bynucleotide insertions, deletions, substitutions, and/or inversions in amanner such that the modifications do not substantially interfere withthe nucleic acid molecule's ability to encode an esterase protein of thepresent invention or to form stable hybrids under stringent conditionswith natural gene isolates.

An arthropod esterase nucleic acid molecule homolog can be producedusing a number of methods known to those skilled in the art (see, forexample, Sambrook et al., ibid.). For example, nucleic acid moleculescan be modified using a variety of techniques including, but not limitedto, classic mutagenesis and recombinant DNA techniques (e.g.,site-directed mutagenesis, chemical treatment, restriction enzymecleavage, ligation of nucleic acid fragments and/or PCR amplification),synthesis of oligonucleotide mixtures and ligation of mixture groups to“build” a mixture of nucleic acid molecules and combinations thereof.Nucleic acid molecule homologs can be selected by hybridization with aC. felis esterase gene or by screening for the function of a proteinencoded by the nucleic acid molecule (e.g., ability to elicit an immuneresponse against at least one epitope of an arthropod esterase protein,hydrolyze α-napthyl acetate, hydrolyze the methyl ester group ofjuvenile hormone and/or bind to DFP).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one arthropod esteraseprotein of the present invention, examples of such proteins beingdisclosed herein. Although the phrase “nucleic acid molecule” primarilyrefers to the physical nucleic acid molecule and the phrase “nucleicacid sequence” primarily refers to the sequence of nucleotides on thenucleic acid molecule, the two phrases can be used interchangeably,especially with respect to a nucleic acid molecule, or a nucleic acidsequence, being capable of encoding an arthropod esterase protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of protecting that animal frominfestation by a hematophagous ectoparasite. As will be disclosed inmore detail below, such a nucleic acid molecule can be, or can encode,an antisense RNA, a molecule capable of triple helix formation, aribozyme, or other nucleic acid-based drug compound. In additionalembodiments, a nucleic acid molecule of the present invention can encodea protective esterase protein (e.g., an esterase protein of the presentinvention), the nucleic acid molecule being delivered to the animal, forexample, by direct injection (i.e, as a naked nucleic acid) or in avehicle such as a recombinant virus vaccine or a recombinant cellvaccine.

One embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE1₄₀₁ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:3.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions ithnucleic acid molecule nfE2₃₆₄ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:4 and/or SEQ ID NO:6.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE3₄₂₁ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:7 and/or SEQ ID NO:9.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE4₅₂₄ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:10 and/or SEQ ID NO:12.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfF5₂₁₄₄ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:57 and/or SEQ ID NO:59.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE6₁₇₉₂ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:18 and/or SEQ ID NO:20.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE7₂₈₃₆ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:24 and/or SEQ ID NO:26.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE8₂₈₀₁ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:30 and/or SEQ ID NO:32.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE9₂₀₀₇ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:36 and/or SEQ ID NO:38.

Another embodiment of the present invention is an esterase nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfE10₁₉₈₇ and preferably with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:67 and/or SEQ ID NO:69.

Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the nucleic acidsequence of nfE1₄₀₁) with nucleic acid sequences reported in GenBankindicates that SEQ ID NO:1 showed no identifiable identity with anysequence reported in GenBank.

Comparison of nucleic acid sequence SEQ ID NO:4 (i.e., the coding strandof nucleic acid sequence of nfE2₃₆₄) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:4 showed the most homolog,i.e., about 43% identity, between SEQ ID NO:4 and a H. virescensjuvenile hormone esterase gene.

Comparison of nucleic acid sequence SEQ ID NO:7 (i.e., the coding strandof nucleic acid sequence of nfE3₄₂₁) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:7 showed the most homolog,i.e., about 53% identity, between SEQ ID NO:7 and a Torpedo marmorataacetylcholinesterase gene.

Comparison of nucleic acid sequence SEQ ID NO:10 (i.e., the codingstrand of nucleic acid sequence of nfE4₅₂₄) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:10 showed the most homolog,i.e., about 47% identity, between SEQ ID NO:10 and an Anas platyrhyncosthioesterase B gene.

Comparison of nucleic acid sequence SEQ ID NO:57 (i.e., the codingstrand of nucleic acid sequence of nfE5₂₁₄₄) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:57 showed the most homolog,i.e., about 41% identity, between SEQ ID NO:57 and a esterase mRNA fromMyzus persicae.

Comparison of nucleic acid sequence SEQ ID NO:18 (i.e., the codingstrand of nucleic acid sequence of nfE6₁₇₉₂) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:18 showed the most homolog,i.e., about 41% identity, between SEQ ID NO:18 and a esterase gene fromMyzus persicae.

Comparison of nucleic acid sequence SEQ ID NO:24 (i.e., the codingstrand of nucleic acid sequence of nfE7₂₈₃₆) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:24 showed the most homolog,i.e., about 48% identity, between SEQ ID NO:24 and an Anas platyrhyncosthioesterase B gene.

Comparison of nucleic acid sequence SEQ ID NO:30 (i.e., the codingstrand of nucleic acid sequence of nfE8₂₈₀₁) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:30 showed the most homolog,i.e., about 46% identity, between SEQ ID NO:30 and a Mus musculuscarboxyl ester lipase gene.

Comparison of nucleic acid sequence SEQ ID NO:36 (i.e., the codingstrand of nucleic acid sequence of nfE9₂₀₀₇) with nucleic acid sequencesreported in GenBank indicates that SEQ ID NO:36 showed the most homolog,i.e., about 47% identity, between SEQ ID NO:36 and a hamster mRNA for CEprecursor gene.

Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:67 showed themost homology, i.e., about 48% identity, between SEQ ID NO:67 and aLucilia cuprina alpha esterase gene (genemb1 #U56636) gene.

Preferred arthropod esterase nucleic acid molecules include nucleic acidmolecules having a nucleic acid sequence that is at least about 55%,preferably at least about 60%, more preferably at least about 65%, morepreferably at least about 70%, more preferably at least about 75%, morepreferably at least about 80%, more preferably at least about 90%, andeven more preferably at least about 95% identical to nucleic acidsequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a proteincomprising amino acid sequence SEQ ID NO:74.

Another preferred nucleic acid molecule of the present intentionincludes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleicacid molecule encoding a protein comprising amino acid sequence SEQ IDNO:74, that is capable of hybridizing to a C. felis esterase gene of thepresent invention, as well as allelic variants thereof. A more preferrednucleic acid molecule includes the nucleic acid sequence SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or anucleic acid molecule encoding a protein comprising amino acid sequenceSEQ ID NO:74, as well as allelic variants thereof. Such nucleic acidmolecules can include nucleotides in addition to those included in theSEQ ID NOs, such as, but not limited to, a full-length gene, afull-length coding region, a nucleic acid molecule encoding a fusionprotein, or a nucleic acid molecule encoding a multivalent protectivecompound. Particularly preferred nucleic acid molecules include nfE1₄₀₁,nfE2₃₆₄, nfE3₄₂₁, nfE4₅₂₄, nfE5₁₉₈₂, nfE5₁₅₁₅, nfE5₂₁₄₄, nfE5₁₆₅₀,nfE6₁₄₈₈, nf6₁₇₉₂, nfE6₁₆₅₀, nfE7₂₈₃₆, nfE7₁₇₈₈, nfE7₁₇₁₀, nfE7₆₅₀,nfE8₂₈₀₁, nfE8₁₇₈₅, nfE8₁₇₁₀, nfE9₂₀₀₇, nfE9₁₅₈₄, nfE9₁₅₄₀, nfE10₁₉₈₇and nfE10₁₅₉₀.

The present invention also includes a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ IDNO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and/or SEQ ID NO:74,including nucleic acid molecules that have been modified to accommodatecodon usage properties of the cells in which such nucleic acid moleculesare to be expressed.

Knowing the nucleic acid sequences of certain arthropod esterase nucleicacid molecules of the present invention allows one skilled in the artto, for example, (a) make copies of those nucleic acid molecules, (b)obtain nucleic acid molecules including at least a portion of suchnucleic acid molecules (e.g., nucleic acid molecules includingfull-length genes, full-length coding regions, regulatory controlsequences, truncated coding regions), and (c) obtain esterase nucleicacid molecules from other arthropods. Such nucleic acid molecules can beobtained in a variety of ways including screening appropriate expressionlibraries with antibodies of the present invention; traditional cloningtechniques using oligonucleotide probes of the present invention toscreen appropriate libraries or DNA; and PCR amplification ofappropriate libraries or DNA using oligonucleotide primers of thepresent invention. Preferred libraries to screen or from which toamplify nucleic acid molecule include flea pre-pupal, 3rd instar oradult cDNA libraries as well as genomic DNA libraries. Similarly,preferred DNA sources to screen or from which to amplify nucleic acidmolecules include flea pre-pupal, 3rd instar or adult cDNA and genomicDNA. Techniques to clone and amplify genes are disclosed, for example,in Sambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingarthropod esterase genes or other arthropod esterase nucleic acidmolecules. Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. Minimal size characteristics are disclosedherein. The present invention includes oligonucleotides that can be usedas, for example, probes to identify nucleic acid molecules, primers toproduce nucleic acid molecules or therapeutic reagents to inhibitesterase protein production or activity (e.g., as antisense-, triplexformation-, ribozyme- and/or RNA drug-based reagents). The presentinvention also includes the use of such oligonucleotides to protectanimals from disease using one or more of such technologies. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto an animal using techniques known to those skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulation of arthropodesterase nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, endoparasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transciption control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rmB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as intermediate early promoters),simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukinis). Transcription controlsequences of the present invention can also include naturally occurringtranscription control sequences naturally associated with arthropods,such as, C. felis.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nfE1₄₀₁, nfE2₃₆₄,nfE3₄₂₁, nfE4₅₂₄, nfE5₁₉₈₂, nfE5₁₅₁₅, nfE5₂₁₄₄, nfE6₁₄₈₈, nfE6₁₇₉₂,nfE6₁₆₅₀, nfE7₂₈₃₆, nfE7₁₇₈₈, nfE7₁₇₁₀, nfE7₆₅₀, nfE8₂₈₀₁, nfE8₁₇₈₅,nfE8₁₇₁₀, nfE9₂₀₀₇, nfE9₁₅₈₄, nfE9₁₅₄₀, nfE10₁₉₈₇ and/or nfE10₁₅₉₀.Particularly preferred recombinant molecules of the present inventioninclude pCro-nfE6₁₄₈₈, pTrc-nfE7₆₅₀, pTrc-nfE7₁₇₁₀, pTrc-nfE8₁₇₁₀,pTrc-nfE5₁₆₅₀, pTrc-nfE9₁₅₄₀, pFB-nfE6₁₆₇₉, pVL-nfE7₁₈₀₂, pVL-fE8₁₇₉₂and pVL-nfE9₁₆₀₀, the production of which are described in the Examplessection.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed arthropod protein of the present invention to besecreted from the cell that produces the protein and/or (b) containfusion sequences which lead to the expression of nucleic acid moleculesof the present invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA),interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments, as well as natural signalsequences. Suitable fusion segments encoded by fusion segment nucleicacids are disclosed herein. In addition, a nucleic acid molecule of thepresent invention can be joined to a fusion segment that directs theencoded protein to the proteosome, such as a ubiquitin fusion segment.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjiection, lipofection, adsorption, andprotoplast fusion. A recombinant cell may remain unicellular or may growinto a tissue, organ or a multicellular organism. Transformed nucleicacid molecules of the present invention can remain extrachromosomal orcan integrate into one or more sites within a chromosome of thetransformed (i.e., recombinant) cell in such a manner that their abilityto be expressed is retained. Preferred nucleic acid molecules with whichto transform a cell include arthropod esterase nucleic acid moleculesdisclosed herein. Particularly preferred nucleic acid molecules withwhich to transform a cell include nfE1₄₀₁, nfE2₃₆₄, nfE3₄₂₁, nfE4₅₂₄,nfE5₁₉₈₂, nfE5₁₅₁₅, nfE5₂₁₄₄, nfE6₁₄₈₈, nfE6₁₇₉₂, nfE6₁₆₅₀, nfE7₂₈₃₆,nfE7₁₇₈₈, nfE7₁₇₁₀, nfE7₆₅₀, nfE8₂₈₀₁, nfE8₁₇₈₅, nfE8₁₇₁₀, nfE9₂₀₀₇,nfE9₁₅₈₄, nfE9₁₅₄₀, nfE10₁₉₈₇ and/or nfE10₁₅₉₀.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing arthropod esterase proteins of thepresent invention or can be capable of producing such proteins afterbeing transformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), parasite, other insect, otheranimal and plant cells. Preferred host cells include bacterial,mycobacterial, yeast, insect and mammalian cells. More preferred hostcells include Salmonella, Escherichia, Bacillus, Listeria,Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamsterkidney) cells, MDCK cells (normal dog kidney cell line for canineherpesvirus cultivation), CRFK cells (normal cat kidney cell line forfeline herpesvirus cultivation), CV-1 cells (African monkey kidney cellline used, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium including attenuated strains such as UK-1_(x)3987 andSR-11_(x)4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCKcells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorgenicmouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriatemammalian cell hosts include other kidney cell lines, other fibroblastcell lines (e.g., human, murine or chicken embryo fibroblast celllines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3cells, LMTK³¹ cells and/or HeLa cells. In one embodiment, the proteinsmay be expressed as heterologous proteins in myeloma cell linesemploying immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed, examples of which aredisclosed herein. Particularly preferred recombinant molecules includepCro-nfE6₁₄₈₈, pTrc-nfE7₆₅₀, pTrc-nfE7₁₇₁₀, pTrc-nfE8₁₇₁₀,pTrc-nfE5₁₆₅₀, pTrc-nfE9₁₅₄₀, pFB-nfE6₁₆₇₉, pVL-nfE7₁₈₀₂, pVL-fE8₁₇₉₂and pVL-nfE9₁₆₀₀.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred a nucleic acid molecules aswell as suitable and preferred recombinant molecules with which totransform cells are disclosed herein. Particularly preferred recombinantcells include E. coli:pCro-nfE6₁₄₈₈ , E. coli:pTrc-nfE7₁₇₁₀ , E.coli:pTrc-nf7₆₅₀ , E. coli:pTrc-nfE8₁₇₁₀ , E. coli:pTrc-nfE5₁₆₅₀ , E.coli:pTrc-nfE9₁₅₄₀ , S. fugiperda:pVL-nfE7₁₈₀₂ , S.fugiperda:pVL-nfE8₁₇₉₂ , S. fugiperda:pVL-nfE9₁₆₀₀ and S.fugiperda:pFB-nfE6₁₆₇₉. Details regarding the production of theserecombinant cells are disclosed herein.

Recombinant cells of the present invention can also be co-transformedwith one or more recombinant molecules including arthropod esterasenucleic acid molecules encoding one or more proteins of the presentinvention and one or more other nucleic acid molecules encoding otherprotective compounds, as disclosed herein (e.g., to produce multivalentvaccines).

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein.

Isolated esterase proteins of the present invention can be produced in avariety of ways, including production and recovery of natural proteins,production and recovery of recombinant proteins, and chemical synthesisof the proteins. In one embodiment, an isolated protein of the presentinvention is produced by culturing a cell capable of expressing theprotein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce an arthropodesterase protein of the present invention. Such medium typicallycomprises an aqueous medium having assimilable carbon, nitrogen andphosphate sources, and appropriate salts, minerals, metals and othernutrients, such as vitamins. Cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes, and petri plates. Culturing can be carried outat a temperature, pH and oxygen content appropriate for a recombinantcell. Such culturing conditions are within the expertise of one ofordinary skill in the art. Examples of suitable conditions are includedin the Examples section.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the perplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein”, as well as similarphrases, refers to collecting the whole fermentation medium containingthe protein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to an arthropodesterase protein of the present invention or a mimetope thereof (i.e.,anti-arthropod esterase antibodies). As used herein, the term“selectively binds to” an esterase protein refers to the ability ofantibodies of the present invention to preferentially bind to specifiedproteins and mimetopes thereof of the present invention. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immiiunioblot assays, etc.; see, forexample, Sambrook et al., ibid. An anti-arthropod esterase antibodypreferably selectively binds to an arthropod esterase protein in such away as to reduce the activity of that protein.

Isolated antibodies of the present invention can include antibodies in abodily fluid (such as, but not limited to, serum), or antibodies thathave been purified to varying degrees. Antibodies of the presentinvention can be polyclonal or monoclonal, functional equivalents suchas antibody fragments and genetically-engineered antibodies, includingsingle chain antibodies or chimeric antibodies that can bind to morethan one epitope.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce arthropod esteraseproteins of the present invention. Antibodies raised against definedproteins or mimetopes can be advantageous because such antibodies arenot substantially contaminated with antibodies against other substancesthat might otherwise cause interference in a diagnostic assay or sideeffects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as therapeutic compounds to passivelyimmunize an animal in order to protect the animal from arthropodssusceptible to treatment by such antibodies and/or (b) as tools toscreen expression libraries and/or to recover desired proteins of thepresent invention from a mixture of proteins and other contaminants.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to hematophagous ectoparasites such as those disclosesherein, in order to directly kill such hematophiagous ectoparasites.Targeting can be accomplished by conjugating (i.e., stably joining) suchantibodies to the cytotoxic agents using techniques known to thoseskilled in the art. Suitable cytotoxic agents are known to those skilledin the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof protecting that animal from infestation by hematophagousectoparasite. Therapeutic compositions of the present invention includeat least one of the following protective compounds: an isolatedhematophagous arthropod esterase protein (including a peptide); amimetope of such a protein; an isolated nucleic acid molecule thathybridizes under stringent hybridization conditions with aCtenocephalides felis esterase gene; an isolated antibody thatselectively binds to an hematophagous arthropod esterase protein; andinhibitors of hematophagous arthropod esterase activity (includingesterase substrate analogs). As used herein, a protective compoundrefers to a compound that, when administered to an animal in aneffective manner, is able to treat, ameliorate, and/or prevent diseasecaused by an arthropod of the present invention. Preferred arthropods totarget are heretofore disclosed. Examples of proteins, nucleic acidmolecules, antibodies and inhibitors of the present invention aredisclosed herein.

A preferred therapeutic composition of the present invention includes atleast one of the following protective compounds: an isolatedhematophagous ectoparasite carboxylesterase protein (including apeptide); a mimetope of such a protein; an isolated hematophagousectoparasite carboxylesterase nucleic acid molecule that hybridizesunder stringent hybridization conditions with a Ctenocephalides feliscarboxylesterase gene; an isolated antibody that selectively binds to ahematophagous ectoparasite carboxylesterase protein; and an inhibitor ofcarboxylesterase activity identified by its ability to inhibit theactivity of a flea carboxylesterase (including a substrate analog).

Suitable inhibitors of esterase activity are compounds that interactdirectly with an esterase protein's active site, thereby inhibiting thatesterase's activity, usually by binding to or otherwise interacting withor otherwise modifying the esterase's active site. Esterase inhibitorscan also interact with other regions of the esterase protein to inhibitesterase activity, for example, by allosteric interaction. Inhibitors ofesterases are usually relatively small compounds and as such differ fromanti-esterase antibodies. Preferably, an esterase inhibitor of thepresent invention is identified by its ability to bind to, or otherwiseinteract with, a flea esterase protein, thereby inhibiting the activityof the flea esterase.

Esterase inhibitors can be used directly as compounds in compositions ofthe present invention to treat animals as long as such compounds are notharmful to host animals being treated. Esterase inhibitors can also beused to identify preferred types of arthropod esterases to target usingcompositions of the present invention, for example by affinitychromatography. Preferred esterase inhibitors of the present inventioninclude, but are not limited to, flea esterase substrate analogs, andother molecules that bind to a flea esterase (e.g., to an allostericsite) in such a manner that esterase activity of the flea esterase isinhibited; examples include, but are not limited to, juvenile hormoneanalogs and cholinesterase inhibitors as well as other neuraltransmission inhibitors. An esterase substrate analog refers to acompound that interacts with (e.g., binds to, associates with, modifies)the active site of an esterase protein. A preferred esterase substrateanalog inhibits esterase activity. Esterase substrate analogs can be ofany inorganic or organic composition, and, as such, can be, but are notlimited to, peptides, nucleic acids, and peptidomimetic compounds.Esterase substrate analogs can be, but need not be, structurally similarto an esterase's natural substrate as long as they can interact with theactive site of that esterase protein. Esterase substrate analogs can bedesigned using computer-generated structures of esterase proteins of thepresent invention or computer structures of esterases' naturalsubstrates. Substrate analogs can also be obtained by generating randomsamples of molecules, such as oligonucleotides, peptides, peptidomimeticcompounds, or other inorganic or organic molecules, and screening suchsamples by affinity chromatography techniques using the correspondingbinding partner, (e.g., a flea esterase). A preferred esterase substrateanalog is a peptidomimetic compound (i.e., a compound that isstructurally and/or functionally similar to a natural substrate of anesterase of the present invention, particularly to the region of thesubstrate that interacts with the esterase active site, but thatinhibits esterase activity upon interacting with the esterase activesite).

Esterase peptides, mimetopes and substrate analogs, as well as otherprotective compounds, can be used directly as compounds in compositionsof the present invention to treat animals as long as such compounds arenot handful to the animals being treated.

The present invention also includes a therapeutic composition comprisingat least one arthropod esterase-based compound of the present inventionin combination with at least one additional compound protective againsthematophagous ectoparasite infestation. Examples of such compounds aredisclosed herein.

In one embodiment, a therapeutic composition of the present inventioncell be used to protect an animal from hematophagous ectoparasiteinfestation by administering such composition to a hematophagousectoparasite, such as to a flea, in order to prevent infestation. Suchadministration could be oral, or by application to the environment(e.g., spraying). Examples of such compositions include, but are notlimited to, transgenic vectors capable of producing at least onetherapeutic composition of the present invention. In another embodiment,a hematophagous ectoparasite, such as a flea, can ingest therapeuticcompositions, or products thereof, present in the blood of a host animalthat has been administered a therapeutic composition of the presentinvention.

Compositions of the present invention can be administered to any animalsusceptible to hematophagous ectoparasite infestation (i.e., a hostanimal), including warm-blooded animals. Preferred animals to treatinclude mammals and birds, with cats, dogs, humans, cattle, chinchillas,ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels,swine, chickens, ostriches, quail and turkeys as well as other furryanimals, pets, zoo animals, work animals and/or food animals, being morepreferred. Particularly preferred animals to protect are cats and dogs.

In accordance with the present invention, a host animal (i.e., an animalthat is or is capable of being infested with a hematophagousectoparasite) is treated by administering to the animal a therapeuticcomposition of the present invention in such a ,manner that thecomposition itself (e.g., an esterase inhibitor, an esterase synthesissuppressor (i.e., a compound that decreases the production of esterasein the hematophagous ectoparasite), an esterase mimetope, or ananti-esterase antibody) or a product generated by the animal in responseto administration of the composition (e.g., antibodies produced inresponse to administration of an arthropod esterase protein or nucleicacid molecule, or conversion of ail inactive inhibitor “prodrug” to anactive esterase inhibitor) ultimately enters the hematophagousectoparasite. A host animal is preferably treated in such a way that thecompound or product thereof enters the blood stream of the animal.Hematophagous ectoparasites are then exposed to the composition orproduct when they feed from the animal. For example, flea esteraseinibitors administered to an animal are administered in such a way thatthe inhibitors enter the blood stream of the animal, where they can betaken up by feeding fleas. In another embodiment, when a host animal isadministered an arthropod esterase protein or nucleic acid molecule, thetreated animal mounts an immune response resulting in the production ofantibodies against the esterase (i.e., anti-esterase antibodies) whichcirculate in the animal's blood stream and are taken up by hematophagousectoparasites upon feeding. Blood taken up by hematophagousectoparasites enters the hematophagous ectoparasites where compounds ofthe present invention, or products thereof, such as anti-esteraseantibodies, esterase inhibitors, esterase mimetopes and/or esterasesynthesis suppressors, interact with, and reduce esterase activity inthe hematophagous ectoparasite.

The present invention also includes the ability to reduce larvalhematophagous ectoparasite infestation in that when hematophagousectoparasites feed from a host animal that has been administered atherapeutic composition of the present invention, at least a portion ofcompounds of the present invention, or products thereof, in the bloodtaken up by the hematophagous ectoparasite are excreted by thehematophagous ectoparasite in feces, which is subsequently ingested byhematophagous ectoparasite larvae. In particular, it is of note thatflea larvae obtain most, if not all, of their nutrition from flea feces.

In accordance with the present invention, reducing esterase activity ina hematophagous ectoparasite can lead to a number of outcomes thatreduce hematophagous ectoparasite burden on treated animals and theirsurrounding environments. Such outcomes include, but are not limited to,(a) reducing the viability of hematophagous ectoparasites that feed fromthe treated animal, (b) reducing the fecundity of female hematophagousectoparasites that feed from the treated animal, (c) reducing thereproductive capacity of male hematophagous ectoparasites that feed fromthe treated animal, (d) reducing the viability of eggs laid by femalehematophagous ectoparasites that feed from the treated animal, (e)altering the blood feeding behavior of hematophagous ectoparasites thatfeed from the treated animal (e.g., hematophagous ectoparasites take upless volume per feeding or feed less frequently), (f) reducing theviability of hematophagous ectoparasite larvae, for example due to thefeeding of larvae from feces of hemator hagous ectoparasites that feedfrom the treated animal and (g) altering the development ofhematophagous ectoparasite larvae (e.g., by decreasing feeding behavior,inhibiting growth, inhibiting (e.g., slowing or blocking) molting,and/or otherwise inhibiting maturation to adults).

Therapeutic compositions of the present invention also includeexcipients in which protective compounds are formulated. An excipientcan be any material that the animal to be treated can tolerate. Examplesof such excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thmerosal or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, dog serum albumin, cat serumalbumin, preservatives, etc., to which sterile water or saline can beadded prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of an animal at a constant rate sufficient to attain therapeuticdose levels of the composition to protect an animal from hematophagousectoparasite infestation. The therapeutic composition is preferablyreleased over a period of time ranging from about 1 to about 12 months.A preferred controlled release formulation of the present invention iscapable of effecting a treatment preferably for at least about 1 month,more preferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

Acceptable protocols to administer therapeutic compositions of thepresent invention in an effective manner include individual dose size,number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art. A suitable single dose is a dose that iscapable of protecting an animal from disease when administered one ormore times over a suitable time period. For example, a preferred singledose of a protein, mimetope or antibody therapeutic composition is fromabout 1 microgram (μg) to about milligrams (mg) of the therapeuticcomposition per kilogram body weight of the animal. Booster vaccinationscan be administered from about 2 weeks to several years after theoriginal administration. Booster administrations preferably areadministered when the immune response of the animal becomes insufficientto protect the animal from disease. A preferred administration scheduleis one in which from about 10 μg to about 1 mg of the therapeuticcomposition per kg body weight of the animal is administered from aboutone to about two times over a time period of from about 2 weeks to about12 months. Modes of administration can include, but are not limited to,subcutaneous, intradermal, intravenous, intranasal, oral, transdermal,intraocular and intramuscular routes.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNAdrug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e., not packaged in a viral coat or cellularmembrane) nucleic acid vaccine (e.g., as naked DNA or RNA molecules,such as is taught, for example in Wolff et al., 1990, Science 247,1465-1468) or (b) administering a nucleic acid molecule packaged as arecombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vechicle).

A naked nucleic acid vaccine of the present invention includes a nucleicacid molecule of the present invention and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidvaccine of the present invention can comprise one or more nucleic acidmolecules of the present invention in the form of, for example, abicistronic recombinant molecule having, for example one or moreinternal ribosome entry sites. Preferred naked nucleic acid vaccinesinclude at least a portion of a viral genome (i.e., a viral vector).Preferred viral vectors include those based on alphaviruses, poxviruses,adenoviruses, herpesviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki virus), species-specificherpesviruses and species-specific poxviruses being particularlypreferred. Any suitable transcription control sequence can be used,including those disclosed as suitable for protein production.Particularly preferred transcription control sequence includecytomegalovirus intermediate early (preferably in conjunction withIntron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specifictranscription control sequences, as well as transcription controlsequences endogenous to viral vectors if viral vectors are used. Theincorporation of “strong” poly(A) sequences are also preferred.

Naked nucleic acid vaccines of the present invention can be administeredin a variety of ways, with intramuscular, subcutaneous, intradermal,transdermal, intranasal and oral routes of administration beingpreferred. A preferred single dose of a naked nucleic acid vaccinesranges from about 1 nanogram (ng) to about 100 μg, depending on theroute of administration and/or method of delivery, as can be determinedby those skilled in the art. Suitable delivery methods include, forexample, by injection, as drops aerosolized and/or topically. Naked DNAof the present invention can be contained in an aqueous excipient (e.g.,phosphate buffered saline) alone or a carrier (e.g., lipid-basedvehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging-deficient and/orencodes an attenuated virus. A number of recombinant viruses can beused, including, but not limited to, those based on alphaviruses,poxviruses, adenoviruses, herpesviruses, and retroviruses. Preferredrecombinant virus vaccines are those based on alphaviruses (such asSindbis virus), raccoon poxviruscs, species-specific herpesviruses andspecies-specific poxviruses. An example of methods to produce and usealphavirus recombinant virus vaccines is disclosed in PCT PublicationNo. WO 94/17813, by Xiong et al., published Aug. 18, 1994, which isincorporated by reference herein in its entirety.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a protective protein or RNA nucleic acid molecule thatis capable of protecting the animal from hematophagous ectoparasiteinfestation. For example, a recombinant virus vaccine comprising anarthropod CE nucleic acid molecule of the present invention isadministered according to a protocol that results in the animalproducing a sufficient immune response to protect itself fromhematophagous ectoparasite infestation. A preferred single dose of arecombinant virus vaccine of the present invention is from about 1×10⁴to about 1×10⁷ virus plaque forming units (pfu) per kilogram body weightof the animal. Administration protocols are similar to those describedherein for protein-based vaccines, with subcutaneous, intramuscular,intranasal and oral administration routes being preferred.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells for this embodimentinclude Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda,yeast, (including Saccharomyces cerevisiae), BHK, CV-1, myoblast G8, COS(e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cellvaccines of the present invention can be administered in a variety ofways but have the advantage that they can be administered orally,preferably at doses ranging from about 10⁸to about 10¹² cells perkilogram body weight. Administration protocols are similar to thosedescribed herein for protein-based vaccines. Recombinant cell vaccinescan comprise whole cells, cells stripped of cell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toprotect an animal from hematophagous ectoparasite infestation can betested in a variety of ways including, but not limited to, detection ofanti-arthropod esterase antibodies (using, for example, proteins ormimetopes of the present invention), detection of cellular immunitywithin the treated animal, or challenge of the treated animal withhematophagous ectoparasites to determine whether, for example, thefeeding, fecundity or viability of hematophagous ectoparasites feedingfrom the treated animal is disrupted. Challenge studies can includeattachment of chambers containing hematophagous ectoparasites onto theskin of the treated animal. In one embodiment, therapeutic compositionscan be tested in animal models such as mice. Such techniques are knownto those skilled in the art.

One preferred embodiment of the present invention is the use ofarthropod protective compounds, such as proteins, mimetopes, nucleicacid molecules, antibodies and inhibitory compounds of the presentinvention, to protect an animal from hematophagous ectoparasite, andparticularly flea, infestation. Preferred protective compounds of thepresent invention include, but are not limited to, C. felis esterasenucleic acid molecules, C. felis esterase proteins and mimetopesthereof, anti-C. felis esterase antibodies, and inhibitors of C. felisesterase activity. More preferred protective compounds of the presentinvention include, but are not limited to, CE or JHE formulations of thepresent invention, C. felis CE nucleic acid molecules, C. felis CEproteins and mimetopes thereof, anti-flea CE antibodies, anti-flea JHEantibodies, inhibitors of C. felis CE activity and inhibitors of fleaJHE activity. Additional protection may be obtained by administeringadditional protective compounds, including other proteins, mimetopes,nucleic acid molecules, antibodies and inhibitory compounds, asdisclosed herein.

One therapeutic composition of the present invention includes aninhibitor of arthropod esterase activity, i.e., a compound capable ofsubstantially interfering with the function of an arthropod esterasesusceptible to inhibition by an inhibitor of arthropod esteraseactivity. An inhibitor of esterase activity can be identified usingarthropod esterase proteins of the present invention. One embodiment ofthe present invention is a method to identify a compound capable ofinhibiting esterase activity of an arthropod. Such a method includes thesteps of (a) contacting (e.g., combining, mixing) an isolated fleaesterase protein, preferably a C. felis esterase protein of the presentinvention, with a putative inhibitory compound under conditions inwhich, in the absence of the compound, the protein has esteraseactivity, and (b) determining if the putative inhibitory compoundinhibits the esterase activity. Putative inhibitory compounds to screeninclude small organic molecules, antibodies (including mimetopesthereof) and substrate analogs. Methods to determine esterase activityare known to those skilled in the art; see, for example, the Examplessection of the present application.

The present invention also includes a test kit to identify a compoundcapable of inhibiting esterase activity of an arthropod. Such a test kitincludes an isolated flea esterase protein, preferably a C. felisesterase protein, having esterase activity and a means for determiningthe extent of inhibition of esterase activity in the presence of (i.e.,effected by) a putative inhibitory compound. Such compounds are alsoscreened to identify those that are substantially not toxic in hostanimals.

Esterase inhibitors isolated by such a method, and/or test kit, can beused to inhibit any esterase that is susceptible to such an inhibitor.Preferred esterase proteins to inhibit are those produced by arthropods.A particularly preferred esterase inhibitor of the present invention iscapable of protecting an animal from hematophagous ectoparasiteinfestation. Effective amounts and dosing regimens can be determinedusing techniques known to those skilled in the art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be known to those skilled in the art. Disclosure of such techniquescan be found, for example, in Sambrook et al., ibid., Borovsky, ArchInsect Biochem. and Phys., 7:187-210, 1988, and related references.

Example 1

This example describes labeling of proteases and esterases withradiolabeled diisopropylfluorophosphate.

Tissue samples were isolated from unfed or bovine blood-fed 1st instarCtenocephalides felis flea larvae; bovine blood-fed or cat blood-fed 3rdinstar Ctenocephalides felis flea larvae; bovine blood-fed or catblood-fed Ctenocephalides felis prepupal flea larvae; bovine blood-fedor cat blood-fed adult Ctenocephalides felis flea midgut tissue, andwhole unfed, bovine blood-fed or cat blood-fed adult Ctenocephalidesfelis fleas. The 1st instar, 3rd instar, prepupal and adult midguttissues were then homogenized by freeze-fracture and sonicated in a Trisbuffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl₂. The whole adultflea sample was then homogenized by freeze-fracture and ground with amicrotube mortar and pestle. The extracts were centrifuged at about14,000×g for 20 minutes (min.) and the soluble material recovered. Thesoluble material was then diluted to a final concentration of about 1 toabout 1.2 tissue equivalents per microliter (μl) of Tris buffer. Eachsample was labeled with [1,3-³H]-diisopropylfluorophosphate (³H-DFP)(available from DuPont-NEN, Wilmington, Del.) using the method generallydescribed in Borovsky, ibid. About 20 tissue equivalents of each tissuesample were mixed with about 1 μCi of ³H-DFP and incubated for about 18hours at 4° C. Proteins contained in each sample were then resolvedusing a 14% Tris-glycine sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) (available from Novex, San Diego, Calif.)under reducing conditions. The gel was soaked in Entensify (availablefrom DuPont-NEN) according to manufacturers instructions, and exposed toX-ray film (available from Kodak X-Omat AR, Rochester, N.Y.) for about 3days at −70° C.

Analysis of the resulting autoradiogram (shown in FIG. 1) indicated thattissue samples from 3rd instar, prepupal larvae and whole adult fleacontained proteins that labeled with DFP, having a molecular weight (MW)of about 60 kilodalton (kD). No proteins of this MW were labeled intissue samples from unfed or fed 1st instar larvae and adult midgut. Theresults indicated preferred tissue distribution and stage-specificexpression of DFP-labeled serine esterases in fleas.

Example 2

This sample describes the identification of general CE activity in fleatissue extracts.

Tissue samples and soluble extracts were prepared as described above inExample 1, except not labelled, from unfed (UF) and bovine blood-fed 1stinstar flea larvae, bovine blood-fed 3rd instar flea larvae, bovineblood-fed prepupal flea larvae, unfed whole adult fleas, cat blood-fedadult (ACF) whole fleas, cat blood-fed adult fleas that have had theirheads and midguts removed (referred to herein as fed adult partialfleas), unfed adult flea midguts and cat blood-fed adult flea midguts.About 5 tissue equivalents of each tissue were assayed for general CEactivity using the following method. Tissue samples of about 5 μl wereadded to separate wells of flat-bottomed microtiter plate (availablefrom Becton Dickinson, Lincoln Park, N.J.). A control well was preparedby adding about 5 μl of Tris buffer to an empty well of the plate. About95 μl of 25 mM Tris-HCl (pH 8.0) was then added to each sample toincrease the volume in each well to about 100 μl. About 100 μl of 0.25mM α-napthyl acetate (available from Sigma, St. Louis, Mo.) dissolved in25 mM Tris-HCl (pH 8.0) was then added to each well. The plate was thenincubated for about 15 min. at 37° C. Following the incubation, about 40μl of 0.3% Fast Blue salt BN (tetrazotized o-dianisidine; available fromSigma) dissolved in 3.3% SDS in water was added to each well.

The microtiter plate was then analyzed using a Cambridge Technology,Inc. (Watertown, Pa.) model 7500 Microplate Reader set to 590 nm. Theabsorbance value for the control sample was subtracted from absorbancevalues of experimental samples, such that the background value was zero.

The results shown in FIG. 2 indicated that general CE activity wasdetected in all tissue samples. The level of activity varied, with unfedand fed 1st instar larvae, unified adult flea midguts, and fed adultflea midguts having relatively lower activity than in the other tissues.Thus, the results indicated preferred tissue distribution andstage-specific expression of general CE activity in fleas.

Example 3

This example describes the determination of general CE activity usingisoelectric focusing (IEF)-PAGE and non-reducing SDS-PAGE.

A. Non-reducing SDS-PAGE.

Soluble extracts from unfed and bovine blood-fed 1st instar flea larvae,bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flealarvae, bovine blood-fed adult (ABF) whole fleas and cat blood-fed adultwhole fleas were prepared using the method described in Example 1. Eachsoluble extract sample was combined with SDS sample buffer (availablefrom Novex) and proteins in the samples were resolved by gelelectrophoresis using 14% Tris-glycine SDS electrophoresis gels(available from Novex). The gels were run at room temperature for about1 hour at 200 volts. After electrophoresis, the gels were soaked forabout for 30 minutes in 50 mM Tris, pH 8.0, containing 2.5% Triton X-100to renature the proteins. The gels were then soaked in 50 mM Tris, pH8.0, for about 5 minutes and then stained for about 5 min. in 50milliliters (ml) of 25 mM Tris, pH 8.0, containing 50 mg Fast blue saltBN and 10 mg α-napthyl acetate (dissolved in 1 ml acetone). Once proteinwas detected on the stained gels, the gels were rinsed with water andphotographed.

B. IEF-PAGE.

Soluble extracts from un fed and bovine blood-fed 1st instar flealarvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fedprepupal flea larvae, unfed and cat blood-fed whole fleas, cat blood-fedadult partial fleas and cat blood-fed adult midguts seere prepared asdescribed above in Section A. The extracts were each combined with IEFsample buffer pH 3-7 (available from Novex) and loaded onto pH 3-7 IEFelectrophoresis gels (available from Novex). The gels wereelectrophoresed at room temperature first for about 1 hour at about 100volts, then for about 1 hour at about 200 volts, and then for about 30min. at about 500 volts. Following electrophoresis, the gels were soakedin 25 mM Tris buffer, pH 8.0, for about 5 min. and then stained forabout 15 min. in 50 ml of 25 mM Tris buffer, pH 8.0, containing 50 mgFast blue salt BN and 10 mg α-napthyl acetate (dissolved in 1 mlacetone). Once protein as detected on the stained gels, the gels wererinsed with water and photographed.

C. Results.

The results from gel electrophoresis experiments described above inSections A and B are shown in FIGS. 3 and 4. The results indicated thatcertain flea tissues contain proteins having MW's of from about 60 toabout 70 kD and native pI values of from about 4.7 to about 5.2 thathave CE activity. In particular, CE activity was identified in prepupallarvae and fed adult flea extracts resolved by non-reduced SDS-PAGE. NoCE activity was identified in unfed and fed 1st instar larvae or fed 3rdinstar larvae extracts (see FIG. 3). When extracts were resolved bynative IEF-PAGE, CE activity was identified in fed 3rd instar larvae,prepupal larvae, unfed and fed whole adult flea, and fed adult partialflea extracts (see FIG. 4, lanes 3-7)). No CE activity was identified inunfed or fed 1st instar larvae, or in fed adult flea midgut extracts(see FIG. 4, lanes 1, 2, and 8).

Example 4

This example describes the purification of CE protein from prepupal flealarvae.

About 15,000 bovine blood-fed prepupal flea larvae were collected andthe larvae were homogenized in TBS by sonication in 50 ml Oak Ridgecentrifuge tubes (available from Nalgene Co., Rochester, N.Y.) bysonicating 4 times 20 seconds each at a setting of 5 of a model W-380Sonicator (available from Heat Systems-Ultrasonics, Inc.). The sonicateswere clarified by centrifugation at 18,000 RPM for 30 minutes to producean extract. Soluble protein in the extract was removed by aspiration anddiluted to a volume of about 20 ml in TBS (equivalent to about 1 larvaper μl TBS). The extract was then added to a column containing about 5ml of p-aminobenzamidine linked to agarose beads (available from Sigma,St. Louis, Mo.) and incubated overnight at 4° C. The column was thenwashed with about 30 ml TBS to remove unbound protein. The collectedunbound protein was then concentrated to a volume of about 20 ml using aMacrosep 10 centrifugal protein concentrator (Filtron Technology Corp.,Northborough Mass.) and filtered sequentially through a 1 μm syringefilter and then through a 0.2 μm syringe filter to clarify the samplefor chromatography.

Aliquots of about 0.5 ml were loaded onto a 20 ml Superdex 200 HR gelfiltration column (available from Pharmacia, Piscataway, N.J.)equilibrated in TBS, operated on a BioLogic liquid chromatography system(available from BioRad, Burlingame, Calif.). About 1 ml fractions werethen collected. Repetitive runs were performed until about 30 ml of eachfraction was collected. The fractions were analyzed for CE activityusing the assay described above in Example 2. In preparation for cationexchange chromatography, fractions having CE activity (V_(e)=16-18 ml)were combined and dialyzed against about 2 liters of 20 mM MES buffer(2-(N-morpholino)ethanesulfonic acid), pH 6.0, containing 10 mM NaCl,for about 1.5 hours, and then against about 1 liter of the same bufferovernight at 4° C. Prior to loading onto the cation exchangechromatography column, the sample was again filtered through a 0.2 μmsyringe filter to remove precipitated proteins. The sample was thenapplied to a Bio-Scale S2 cation exchange column (available from BioRad)at a rate of about 0.5 ml/min. The column was washed with MES bufferuntil all unbound protein was removed. Protein bound to the column wasthen eluted with a linear gradient from 10 mM to 1 M NaCl in 20 mM MESbuffer, pH 6. Fractions were assayed for CE activity using the assaydescribed above in Example 2. The results indicated that CE activity wasnot retained on the cation exchange column using the above conditions,and all of the activity was found in the flow-through fractions.

Fractions containing CE activity were pooled and adjusted to pH 7 using0.5 M Tris, pH 8.0, in preparation for anion exchange chromatography.The pooled fractions were then loaded onto a 4.5 mm×50 mm Poros 10 HQanion exchange chromatography column (available from PerSeptiveBiosystems, Cambridge, Mass.) equilibrated in 25 mM Tris buffer, pH 6.8.The column was washed with the loading buffer, and bound proteins wereeluted with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH6.8. Fractions were tested for CE activity using the assay describedabove in Example 2. The results indicated that CE activity was eluted atabout 170 mM NaCl. Fractions containing CE activity were pooled anddiafiltered into TBS.

Example 5

This example describes the determination of N-terminal amino acidsequences of carboxylesterases isolated from prepupal flea larvae.

A. Anion Exchange Chromatography Fractions.

Anion exchange chromatography fractions described above in Example 4that contained proteins having CE activity were pooled, diafiltered intoTBS buffer and concentrated 3-fold in a Speed-Vac Concentrator(available from Savant Instruments, Holbrook, N.Y.). Proteins in theconcentrated samples were then resolved on a reducing, 10% SDS-PAGETris-glycine gel (available from Novex) for 1 hour at about 200 V. Theproteins on the gel were then blotted onto a polyvinylidene difluoride(PVDF) membrane (available from Novex) for about 70 min in 10 mM CAPSbuffer (3-[cyclohexylamino]-1-propanesulfonic acid; available fromSigma), pH 11, with 0.5 mM dithiothreitol (DTT). The membrane was thenstained for 1 minute in 0.1% Coomassie Blue R-250 dissolved in 40%methanol and 1% acetic acid. The membrane was destained in 50% methanolfor about 10 minutes, rinsed with MilliQ water and air dried. Threestained protein bands were identified having apparent molecular weightsof about 64 kD, 65 kD, and 66 kD, respectively. The portion of themembrane containing each band was excised separately. Protein containedin each membrane segment was subjected to N-terminal amino acidsequencing using a 473A Protein Sequencer (available from AppliedBiosystems, Foster City, Calif.) and using standard techniques.

The results indicated that the N-terminal amino acid sequence of theputative 64 kD protein was DPPTVTLPQGEL (denoted SEQ ID NO:39); theN-terminal amino acid sequence of the putative 65 kD protein wasDPPTVTLPQGELVGKATNEnxk (denoted SEQ ID NO:40); and the N-terminal aminoacid sequence of the putative 66 kD protein was DppTVTLPQGEL (denotedSEQ ID NO:41), in which the lower case letters designate uncertaintiesand “x” designates an undetermined residue.

B. Proteins Resolved by Native IEF-PAGE.

Proteins isolated by anion exchange chromatography as described above inSection A were further resolved by native IEF-PAGE. Proteins were loadedonto a pH 3-10 IEF gel (available from Novex) and separated in Novex'sIEF buffers according to Novex's standard procedure (60 min at 100 V;then 60 min at 200 V; and then 30 min at 500 V). Followingelectrophoresis, part of the gel was stained for CE activity using themethod described above in Example 2. The remaining portion of the gelwas blotted onto PVDF membrane by reversing the orientation of the geland membrane so that positively charged proteins migrated to themembrane, electrophoresing the protein for 60 min at 10 V, using 0.7%acetic acid as the transfer buffer. The membrane was stained asdescribed above in Section A. After the membrane was dried, stainedprotein bands on the membrane were compared to bands on the gel testedfor CE activity to identify corresponding bands. Protein bands on themembrane corresponding to proteins having CE activity were excised andsubmitted to N-terminal sequencing as described in Section A.

N-terminal amino acid sequence was obtained for protein contained in twobands having pI values of about pI 4.8 and about pI 4.9. N-terminalamino acid sequence of the pI 4.8 band was DPPTVTLPQGELVGKALSNen(denoted SEQ ID NO:42) and N-terminal amino acid sequence of the pI 4.9band was DPPTVTLP (denoted SEQ ID NO:43). A comparison of the N-terminalamino acid sequences identified here and described in Section Aindicates closely related proteins having a consensus sequence ofDPPTVTLPQGELVGKALTNEnGk (denoted SEQ ID NO:44).

The amino acid sequences of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 are substantially containedwithin SEQ ID NO:5, SEQ ID NO:19 and SEQ ID NO:53, which are describedbelow in Example 11.

Example 6

This example describes partial purification of CE from 3rd instar flealarvae.

Using the extract preparation methods described in Example 1 withoutlabelling, extracts were prepared from about 50,000 bovine blood-fed 3rdinstar flea larvae. The extract was then further purified over ap-aminobenzamidine linked agarose bead column using the method alsodescribed in Example 1. Collected unbound protein was concentrated toabout 70 ml using a 200 ml stirred cell fitted with a YM-10 membrane(available from Amicon, Beverly, Mass.). Seven ml (about 5,000 3rdinstar flea larval equivalents) of the concentrated extract was used forthe remainder of the purification scheme described in Example 4.Resulting fractions from the anion exchange chromatography column weretested for CE activity using the assay described above in Example 2.

The results indicated that CE activity was eluted in two overlappingpeaks at about 120 mM and about 210 mM NaCl.

Example 7

This example describes the identification of JHE activity in differentflea tissues.

Tissue samples were prepared as described above in Example 1 from unfedand bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instarflea larvae, bovine blood-fed prepupal flea larvae, unfed and catblood-fed whole adult fleas, cat blood-fed adult partial fleas and catblood-fed adult flea midguts. About 5 tissue equivalents of each tissuewas assayed for JHE activity as follows.

Unlabeled juvenile hormone (JH; available from ICN Biomedicals, Inc.,Aurora, Ohio) was diluted in hexane to concentration of about 0.025 M.Labeled 10-³H-juvenile hormone (³H-JH; available from Dupont-NEN) wasdiluted in hexane to concentration of about 80,000 cpm/μl. A JHsubstrate mixture was prepared by mixing about 20 μl of unlabeled JHwith about 80 μl of ³H-JH (about 5 μCi) in a 4 ml screw cap vial. Thesubstrate mixture was then covered with nitrogen (i.e., “blanketed”) andthe solvent contained in the mixture was evaporated by heating themixture at 35° C. When just dry, about 1 ml of absolute anhydrousethanol (final concentration 5×10⁻⁴ M, or 6400 cpm/μl) was added to thevial. The substrate mixture was then stored at −20° C.

About 5 equivalents of each tissue (about 5 μl of protein) was addedinto the bottom of a small glass autosampler vial. About 95 μl ofTris-buffered saline (TBS) was added to each vial to bring the finalvolume in each vial to about 100 μl. Two control samples were alsoprepared by adding 100 μl TBS to two separate vials. About 1 μl of thesubstrate mixture described above was added to all of the vialsincluding the control samples. The final JH concentration in each vialwas about 5×10⁻⁶ M. The vials were then capped and spun in a microfugeto bring all of the liquid to the bottom of each vial. The vials werethen transferred to a heat block and incubated at 35° C. for about 30minutes. Following the incubation, enzyme activity was stopped by addingabout 50 μl of methanol buffer (methanol:water:concentrated ammoniumhydroxide at a 10:9:1 ratio, respectively) to each vial and removing thevials from the heat block.

To measure labeled juvenile hormone acid, about 250 μl isooctane wasadded to each vial. Each vial was vortexed for about 15 seconds or untilan emulsion formed. Each vial was then centrifuged in a microfuge forabout 1 minute to separate aqueous and organic phases. About 75 μl ofthe aqueous layer was removed from each vial and added to about 2 nlEco-lame scintillation fluid (available from ICN). The amount of³H-juvenile hormone acid contained in each vial was determined using aBeckman LS-1801 liquid scintillation counter (available from Beckman,Fullerton, Calif.).

The results shown in FIG. 5 indicated that all flea tissues testedcontain active JHE. Referring to Example 2, the level of CE activitydiffered from JHE activity in various tissue samples. The combined JHEand CE data indicated the differential expression of these two enzymaticactivities during the development of a flea.

Example 8

This example describes the purification of JHE protein from catblood-fed adult midguts.

About 23,000 cat blood-fed adult midguts were collected and preparedusing the method described in Example 1. The extract was then added in 4aliquots to columns containing about 3 to about 5 ml ofp-aminobenzamidine linked agarose beads (available from Sigma),equilibrated in 50 mM Tris (pH 8.0), 100 mM CaCl₂, 400 mM NaCl, andincubated overnight at 4° C. The columns were then washed with about 15to about 125 ml of the equilibration Tris buffer to removes unboundprotein. The collected unbound protein was pooled and then concentratedto a volume of about 5 ml using an Ultrafree-20 10 kD centrifugalconcentrator (available from Millipore, Bedford, Mass.) and filteredsequentially through a 0.2 μm centrifugal ultrafiltration membrane(available from Lida, Kenosha, Wis.) to clarify the sample forchromatography.

Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gelfiltration column using the method described in Example 4. Repeated runswere performed until about 10 ml of each fraction was collected. Thefractions were analyzed for JHE activity using the assay described inExample 7. In preparation for anion exchange chromatography, fractionshaving JHE activity (V_(e)=17-18 ml) were combined and dialyzedovernight against about 1 L of 20 mM Tris buffer, pH 8.0, containing 10mM NaCl. The sample was then loaded onto a Poros 10 HQ anion exchangecolumn using the method described in Example 4. Resulting fractions weretested for JHE activity as described in Example 7.

The results indicated that midgut JHE activity was eluted from the anionexchange column in a single peak at about 120 mM NaCl.

Example 9

This example describes partial purification of JHE from prepupal flealarvae and 3rd instar larvae.

A. JHE Purification From Prepupal Tissue.

Using the extract preparation methods described in Example 1, gelfiltration fractions were obtained using a Superdex 200 HR gelfiltration column (available from Pharmacia) using the method describedin Example 4, from about 15,000 bovine blood-fed prepupal flea larvae.The fractions were analyzed for JHE activity using the assay, describedabove in Example 7. Those fractions containing protein having JHEactivity (V_(e)=16-18 ml) were combined and dialyzed using the methoddescribed in Example 8.

The fractions were then further purified by passing the fractions over aBio-Scale S2 cation exchange column (available from BioRad) at a rate ofabout 0.5 ml/min. The column was washed with MES until all unboundprotein was eluted. Bound protein was then eluted with a linear gradientof 20 mM MES buffer, pH 6.0, containing 10 mM NaCl to 1 M NaCl.Resulting fractions were assayed for JHE activity using the methoddescribed in Example 7. The results indicated that proteins having JHEactivity using prepupal tissue eluted from the column in about 200 to300 mM NaCl.

The fractions containing JHE activity were combined and the pH adjustedto pH 7 using 0.5 M Tris buffer (pH 8.0). The fractions were thendialyzed twice against about 1 liter of 10 mM phosphate buffer (pH 7.2)containing 10 mM NaCl at 4° C. The resulting dialyzed fractions werethen loaded onto a Bio-Scale CHT2-I Hydroxyapatite Column (availablefrom BioRad) at a rate of about 0.5 ml/min. Unbound protein was washedfrom the column using the dialysis buffer. Bound protein was then elutedwith a linear gradient of from 10 mM phosphate buffer, pH 7.2,containing 10 mM NaCl to 0.5 M phosphate buffer pH 6.5 containing 10 mMNaCl. One ml fractions were collected and each tested for JHE activityby the method described in Example 7.

The results indicated that JHE eluted in 2 overlapping peaks at about100 mM and 150 mM phosphate. These two JHE activities were designated PPJHE I and PP JHE II, and were kept separate for the remainder of thepurification. Both JHE samples, were dialyzed overnight against 20 mMTris buffer (pH 8.0) containing 10 mM NaCl. The two samples were thenloaded, separately, onto a 4.5 mm×50 mm Poros 10 HQ anion exchangechromatography column (available from PerSeptive Biosystems)equilibrated with 20 mM Tris buffer, pH 8.0, containing 10 mM NaCl.Unbound proteins were washed from the column using the same buffer.Bound proteins were eluted with a linear gradient of from 10 mM to 1 MNaCl in 20 mM Tris buffer, pH 8.0. Resulting fractions were tested forJHE activity using the method described in Example 7.

The results indicated that in both samples, JHE activity was eluted fromthe column in a single peak at about 100 mM NaCl.

B. JHE Purification From 3rd Instar Tissue

Using the procedure described above in Section A, proteins having JHEactivity were obtained using about 5,000 bovine blood-fed 3rd instarflea larvae. Following purification by cation exchange, proteins havingJHE activity using 3rd instar tissue were found to elute in 2 peaks. Thefirst peak having JHE activity was not retained on the column and alsoexhibited CE activity (referred to herein as CE/JHE fractions). Thesecond peak having JHE activity eluted from the column in about 100-200mM NaCl and did not contain CE activity.

The CE/JHE fractions were pooled and adjusted to about pH 7 using 0.5 MTris, pH 8.0. The fractions were then loaded onto a 4.5 mm×50 mm Poros10 HQ anion exchange chromatography column (available from PerSeptiveBiosystems) and the column was equilibrated in 25 mM Tris buffer, pH6.8. The column was washed with the same buffer and bound proteins wereeluted with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH6.8. Fractions were then tested for JHE activity using the methoddescribed in Example 7. JHE activity was eluted in two overlapping peaksat about 120 mM and 210 mM NaCl. The fraction containing JHE activityalso contained CE activity when tested using the method described inExample 2.

Fractions from the cation exchange column containing only JHE activitywere combined, diluted in 20 mM Tris buffer, pH 8 containing 10 mM NaCl,and concentrated to about 5 ml. The fractions were purified on a Poros10 HQ anion exchange chromatography column as described immediatelyabove. Fractions were then tested for JHE activity using the methoddescribed in Example 7. The JHE activity was eluted in a single peak atabout 120 mM. The peak contained no detectable CE activity.

Example 10

This example describes the purification of JHE protein from unfed adultmidguts.

About 16,000 unfed adult midguts were collected in 20 mM Tris buffer (pH7.7), containing 130 mM NaCl, 1 mM sodium EDTA, 1 mM Pefabloc®(available from Boehringer Mannheim, Indianapolis, Ind.), 1 microgram/ml(μg/ml) leupeptin and 1 μg/ml pepstatin. The midguts were homogenized byfreeze-fracture and sonication, and then centrifuged at about 14,000×gfor 20 min. The soluble material from the centrifugation step wasrecovered. The soluble material was then concentrated to about 1 mlusing an Ultrafree-20 10 kD centrifugal concentrator (available fromMillipore) and filtered sequentially through a 0.2 μm centrifugalultrafiltration membrane to clarify the sample for chromatography.Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR gelfiltration column using the method described in Example 4. Repeatedcolumn runs were performed until about 2 ml of each fraction wascollected. The fractions were analyzed for JHE activity using the assaydescribed in Example 7. In preparation for cation exchangechromatography, fractions having JHE activity (V_(c)=15-17 ml) werecombined and dialyzed overnight against about 1 L of 20 mM MES buffer,pH 6.0, containing 10 mM NaCl. The sample was then applied to aBio-Scale S2 cation exchange column using the method described inExample 4. Fractions of eluate were assayed for JHE activity using themethod described in Example 7.

The results indicate that JHE is present in unfed midguts in two forms,one that is not retained on the cation exchange column and one that isbound to the column under low salt conditions at about 100 mM NaCl. Theform that was not retained under low salt conditions was shown to havegeneral CE activity using the methods described in Example 2.

Example 11

This example describes the identification of certain esterase nucleicacid molecules of the present invention.

Several flea esterase nucleic acid molecules, representing one or morepartial flea esterase genes, were PCR amplified from a flea mixed instarcDNA library or a flea prepupal cDNA library. The flea mixed instar cDNAlibrary was produced using unfed 1st instar, bovine blood-fed 1stinstar, bovine blood-fed 2nd instar and bovine blood-fed 3rd instar flealarvae (this combination of tissues is referred to herein as mixedinstar larval tissues for purposes of this example). The flea prepupalcDNA library was produced using prepupal flea larvae. For each library,total RNA was extracted from mixed instar or prepupal tissue,respectfully, using an acid-guanidinium-phenol-chloroform method similarto that described by Chomczynski et al., 1987, Anal. Biochem. 162, p.156-159. Approximately 5,164 mixed instar larvae or 3,653 prepupallarvae were used in each RNA preparation. Poly A+ selected RNA wasseparated from each total RNA preparation by oligo-dT cellulosechromatography using Poly(A)Quick® mRNA isolation kits (available fromStratagene Cloning Systems, La Jolla, Calif.), according to the methodrecommended by the manufacturer.

A mixed instar cDNA expression library and a prepupal cDNA expressionlibrary were constructed in lambda (λ) Uni-ZAP™XR vector (available fromStratagene Cloning Systems) using Stratagene's ZAP-cDNA Synthesis Kit®protocol. About 6.34 μg of mixed instar poly A+ RNA were used to producethe mixed instar library and about 6.72 μg of prepupal poly A+ RNA wereused to produce the prepupal library. The resultant mixed instar librarywas amplified to a titer of about 2.17×10¹⁰ pfu/ml with about 97%recombinants. The resultant prepupal library was amplified to a titer ofabout 3.5×10¹⁰ pfu/ml with about 97% recombinants.

A pair of primers was used to amplify DNA from the cDNA libraries. Asense vector primer T-3X (corresponding to the vector in which nucleicacid molecules of the present invention had been ligated), having thenucleic acid sequence AATTAACCCT CACTAAAGGG (available from Gibco BRL,Gaithersburg, Md.; denoted SEQ ID NO:45), was used in combination with adegenerate primer, the design of which was based on a highly conservedesterase amino acid sequence (disclosed in Hanzlik et al., J. Biol.Chem. 264:12419-12423, 1989; I Y/H G G G F/L) located in a regiondownstream from the mature amino terminus in a number of knownesterases. The degenerate primer, referred to herein as FCEF, is ananti-sense primer having the nucleic acid sequence ARDCCDCCDC CRTRDAT (Rindicating an A or G; and D indicating an A, G or T; denoted SEQ IDNO:46). The resultant PCR products from the mixed instar cDNA library,obtained using standard PCR conditions (e.g., Sambrook et al., ibid.).were about 550 nucleotides. The resultant PCR products from the prepupalcDNA library were from about 500 nucleotides to about 860 nucleotides.

A. PCR Products.

PCR products were gel purified and cloned into the TA Vector™ (availablefrom InVitrogen Corp., San Diego, Calif.). Approximately 8 clones wereidentified from the prepupal library and 6 clones were identified fromthe mixed instar library. These nucleic acid molecules were subjected tonucleic acid sequencing using the Sanger dideoxy chain terminationmethod, as described in Sambrook et al., ibid.

1. Flea esterase clone 1 isolated from the mixed instar cDNA library wasdetermined to comprise nucleic acid molecule nfE1₄₀₁, the nucleic acidsequence of the coding strand which is denoted herein as SEQ ID NO:1.Translation of SEQ ID NO:1 suggests that nucleic acid molecule nfE1₄₀₁encodes a non-full-length flea esterase protein of about 103 aminoacids, referred to herein as PfE1₁₀₃, having amino acid sequence SEQ IDNO:2, assuming an initiation codon spanning from nucleotide 92 throughnucleotide 94 of SEQ ID NO:1. The complement of SEQ ID NO:1 isrepresented herein by SEQ ID NO:3. Comparison of amino acid sequence SEQID NO:2 (i.e., the amino acid sequence of PfE1₁₀₃) with amino acidsequences reported in GenBank indicates that SEQ ID NO:2, showed themost homology, i.e., about 33% identity, between SEQ ID NO:2 and alphaesterase protein from Drosophila melanogaster.

2. Flea esterase clone 2 isolated from the mixed instar cDNA library wasdetermined to comprise nucleic acid molecule nfE2₃₆₄, the nucleic acidsequence of the coding strand which is denoted herein as SEQ ID NO:4.Translation of SEQ ID NO:4 suggests that nucleic acid molecule nfE2₃₆₄encodes a non-full-length flea esterase protein of about 121 aminoacids, referred to herein as PfE₁₂₁, having amino acid sequence SEQ IDNO:5, assuming the first codon spans from nucleotide 2 throughnucleotide 4 of SEQ ID NO:4. The complement of SEQ ID NO:4 isrepresented herein by SEQ ID NO:6. Comparison of nucleic acid sequenceSEQ ID NO:4 with nucleic acid sequences reported in GenBank indicatesthat SEQ ID NO:4 showed the most homology, i.e., about 43% identity,between SEQ ID NO:4 and a H. virescens JHE gene. Comparison of aminoacid sequence SEQ ID NO:5 (i.e., the amino acid sequence of PfE2₁₂₁)with amino acid sequences reported in GenBank indicates that SEQ IDNO:5, showed the most homology, i.e., about 38% identity, between SEQ IDNO:5 and alpha esterase protein from Drosophila melanogaster.

3. Flea esterase clone 3 isolated from the prepupal cDNA library wasdetermined to comprise nucleic acid molecule nfE3₄₂₁, the nucleic acidsequence of the coding strand which is denoted herein as SEQ ID NO:7.Translation of SEQ ID NO:7 suggests that nucleic acid molecule nfE3₄₂₁encodes a non-full-length flea esterase protein of about 103 aminoacids, referred to herein as PfE3₁₀₃, hatting amino acid sequence SEQ IDNO:8, assuming an initiation codon spanning from nucleotide 113 throughnucleotide 115 of SEQ ID NO:7. The complement of SEQ ID NO:7 isrepresented herein by SEQ ID NO:9. Comparison of nucleic acid sequenceSEQ ID NO:7 with nucleic acid sequences reported in GenBank indicatesthat SEQ ID NO:7 showed the most homology, i.e., about 53% identity,between SEQ ID NO:7 and a Torpedo marmorata acetylcholinesterase gene.Comparison of amino acid sequence SEQ ID NO:8 (i.e., the amino acidsequence of PfE3₁₀₃) with amino acid sequences reported in GenBankindicates that SEQ ID NO:8, showed the most homology, i.e., about 39%identity, between SEQ ID NO:5 and alpha esterase protein from Drosophilamelanogaster.

4. Flea esterase clone 4 isolated from the prepupal cDNA library wasdetermined to comprise nucleic acid molecule nfE4₅₂₄, the nucleic acidsequence of the coding strand which is denoted herein as SEQ ID NO:10.Translation of SEQ ID NO:10 suggests that nucleic acid molecule nfE4₅₂₄encodes a non-full-length flea esterase protein of about 137 aminoacids, referred to herein as PfE4₁₃₇, having amino acid sequence SEQ IDNO:11, assuming an initiation codon spanning from nucleotide 113 throughnucleotide 115 of SEQ ID NO:10. The complement of SEQ ID NO:10 isrepresented herein by SEQ ID NO:12. Comparison of nucleic acid sequenceSEQ ID NO:10 with nucleic acid sequences reported in GenBank indicatesthat SEQ ID NO:10 showed the most homology, i.e., about 47% identity,between SEQ ID NO:10 and an Anas platyrhyncos thioesterase B gene.Comparison of amino acid sequence SEQ ID NO:11 (i.e., the amino acidsequence of PfE4₁₃₇) with amino acid sequences reported in GenBankindicates that SEQ ID NO:11, showed the most homology, i.e., about 30%identity, between SEQ ID NO:11 and Leptinotarsa decemlineataacetylcholinesterase.

B. cDNA Clones.

Certain amplified PCR fragments were used as probes to identifyfull-length flea esterase genes in the prepupal cDNA library.

1. Nucleic acid molecule nfE2₃₆₄ was labeled with ³²P and used as aprobe to screen the mixed instar cDNA library described in Section A,using standard hybridization techniques. Two clones were isolated. Afirst clone included about a 2300-nucleotide insert, referred to hereinas nfE5₂₃₀₀. Nucleic acid sequence was obtained using standardtechniques from nfE5₂₃₀₀, to yield a flea esterase nucleic acid moleculenamed nfE5₁₉₈₂ having a nucleic acid sequence of the coding strand whichis denoted herein as SEQ ID NO:13. Translation of SEQ ID NO:13 suggeststhat nucleic acid molecule nfE5₁₉₈₂ encodes a non-full-length fleaesterase protein of about 505 amino acids, referred to herein asPfE5₅₀₅, having amino acid sequence SEQ ID NO:14, assuming the firstcodon spans from nucleotide 1 through nucleotide 3 of SEQ ID NO:13 andthe stop codon spans from nucleotide 1518 through nucleotide 1520 of SEQID NO:13. The complement of SEQ ID NO:13 is represented herein by SEQ IDNO:15. The amino acid sequence of PfE5₅₀₅ (i.e., SEQ ID NO:14) predictsthat PfE5₅₀₅ has an estimated molecular weight of about 56.8 kD and anestimated pI of about 5.5. Tile nucleic acid molecule representing thecoding region for PfE5₅₀₅ is referred to herein as nfE5₅₁₅; the nucleicacid sequences of the coding strand and the complementary strand arerepresented by SEQ ID NO:16 and SEQ ID NO:17, respectively.

The nucleic acid sequence of nfE5₁₉₈₂ was used to design primers to usein combination with a vector primer to PCR amplify the 5′ terminalfragment of the remainder of the flea esterase coding region from theflea mixed instar cDNA library. A pair of primers was used to amplifyDNA from the cDNA library. A sense vector primer T3-X (corresponding tothe vector in which nucleic acid molecules of the present invention hadbeen ligated), having the nucleic acid sequence 5′ AATTAACCCT CACTAAAGGG3′ (denoted SEQ ID NO:45), was used in combination with an anti-senseprimer M6/M265′, having the nucleic acid sequence 5′ GTGCGTACACGTTTACTACC 3′ (denoted SEQ ID NO:56). The resultant PCR product from themixed instar cDNA library, obtained using standard PCR conditions (e.g.,Sambrook et al., ibid.), were about 354 nucleotides.

The PCR product was subjected to DNA sequencing analysis, and acomposite sequence representing a full-length flea esterase codingregion was deduced. The nucleic acid sequence of the composite nucleicacid molecule, referred to herein as nfE5₂₁₄₄ is denoted herein as SEQID NO:57. Translation of SEQ ID NO:57 suggests that nucleic acidmolecule nfE5₂₁₄₄ encodes a full-length flea esterase protein of about550 amino acids, referred to herein as PfE5₅₅₀, having amino acidsequence SEQ ID NO:58, assuming an open reading frame in which theinitiation codon spans from nucleotide 30 through nucleotide 32 of SEQID NO:57 and the stop codon spans from nucleotide 1680 throughnucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 isrepresented herein by SEQ ID NO:59. The coding region encoding PfE5₅₅₀is represented by the nucleic acid molecule nfE5₁₆₅₀, having a codingstrand with the nucleic acid sequence represented by SEQ ID NO:60 and acomplementary strand with nucleic acid sequence SEQ ID NO:61. The aminoacid sequence of PfE5₅₅₀ (i.e., SEQ ID NO:58) predicts that PfE5₅₅₀ hasan estimated molecular weight of about 61.8 kD and an estimated pI ofabout 5.5.

Comparison of nucleic acid sequence SEQ ID NO:57 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:57 showed themost homology, i.e., about 41% identity, between SEQ ID NO:57 and a M.persicae esterase FE4 mRNA sequence. Comparison of amino acid sequenceSEQ ID NO:58 (i.e., the amino acid sequence of PfE5₅₅₀) with amino acidsequences reported in GenBank indicates that SEQ ID NO:58 showed themost homology, i.e., about 36% identity between SEQ ID NO:58 andDrosophila melanogaster alpha esterase protein.

A second clone included about a 1900 nucleotide insert, referred toherein as nfE6₁₉₀₀. Nucleic acid sequence was obtained using standardtechniques from nfE6₁₉₀₀, to yield a flea esterase nucleic acid moleculenamed nfE6₁₇₉₂ having a nucleic acid sequence of the coding strand whichis denoted herein as SEQ ID NO:18. Translation of SEQ ID NO:18 suggeststhat nucleic acid molecule nfE6₁₇₉₂ encodes a full-length flea esteraseprotein of about 550 amino acids, referred to herein as PfE6₅₅₀, havingamino acid sequence SEQ ID NO:19, assuming an open reading frame inwhich the initiation codon spans from nucleotide 49 through nucleotide51 of SEQ ID NO:18 and a stop codon spanning from nucleotide 1699through nucleotide 1701 of SEQ ID NO:18. The complement of SEQ ID NO:18is represented herein by SEQ ID NO:20. The coding region encodingPfE6₅₅₁, is represented by nucleic acid molecule nfE6₁₆₅₀, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:21and a complementary strand with nucleic acid sequence SEQ ID NO:22. Theproposed mature protein, denoted herein as PfE6₅₃₀, contains about 530amino acids which is represented herein as SEQ ID NO:53. The nucleicacid molecule encoding PfE6₅₃₀ is denoted herein as nfE6₁₅₉₀ and has acoding strand having the nucleic acid sequence SEQ ID NO:23. The aminoacid sequence of PfE6₅₅₀ (i.e., SEQ ID NO:19) predicts that PfE6₅₅₀ hasan estimated molecular weight of about 61.8 kD and an estimated pI ofabout 5.5.

Comparison of nucleic acid sequence SEQ ID NO:18 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:18 showed themost homology, i.e., about 41% identity, between SEQ ID NO:18 and aMyzus pericae esterase gene. Comparison of amino acid sequence SEQ IDNO:19 (i.e., the amino acid sequence of PfE6₅₅₀) with amino acidsequences reported in GenBank indicates that SEQ ID NO:19 showed themost homology, i.e., about 28% identity between SEQ ID NO:19 andDrosophila melanogaster alpha esterase protein.

2. Nucleic acid molecule nfE4₁₂₄ was labeled with ³²P and used as aprobe to screen the prepupal cDNA library described in Example 11, usingstandard hybridization techniques (e.g., Sambrook et al., ibid.). Twoclones were isolated. A first clone included about a 3000 nucleotideinsert, referred to herein as nfE7₃₀₀₀. Nucleic acid sequence wasobtained using standard techniques from nfE7₃₀₀₀, to yield a fleaesterase nucleic acid molecule named nfE7₂₈₃₆ having a nucleic acidsequence of the coding strand which is denoted herein as SEQ ID NO:24.Translation of SEQ ID NO:24 suggests that nucleic acid molecule nfE7₂8₃₆encodes a full-length flea esterase protein of about 596 amino acids,referred to herein as PfE7₁₉₆, having amino acid sequence SEQ ID NO:25,assuming an open reading frame in which the initiation codon spans fromnucleotide 99 through nucleotide 101 of SEQ ID NO:24 and a stop codonspanning from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:25.The complement of SEQ ID NO:24 is represented herein by SEQ ID NO:26.The coding region encoding PfE7₅₉₆, is represented by nucleic acidmolecule nfE7₁₇₈₈, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:28 and a complementary strand with nucleic acidsequence SEQ ID NO:29. The proposed mature protein, denoted herein asPfE7₅₇₀, contains about 570 amino acids which is represented herein asSEQ ID NO:54. The nucleic acid molecule encoding PfE7₅₇₀ is denotedherein as nfE7₁₇₁₀ and has a coding strand having the nucleic acidsequence SEQ ID NO:27. The amino acid sequence of PfE7₅₉₆ (i.e., SEQ IDNO:25) predicts that PfE7₅₉₆ has an estimated molecular weight of about68.7 kD and an estimated pI of about 6.1.

Comparison of nucleic acid sequence SEQ ID NO:24 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:24 showed themost homology, i.e., about 48% identity, between SEQ ID NO:24 and anAnas platyrhyncos thioesterase B gene. Comparison of amino acid sequenceSEQ ID NO:25 (i.e., the amino acid sequence of PfE7₅₉₆) With amino acidsequences reported in GenBank indicates that SEQ ID NO:25 showed themost homology, i.e., about 27% identity between SEQ ID NO:25 andDrosophila melanogaster alpha esterase protein.

A second clone included about a 3000 nucleotide insert, referred toherein as nfE8₃₀₀₀. Nucleic acid sequence was obtained using standardtechniques from nfE8₃₀₀₀, to yield a flea esterase nucleic acid moleculenamed nfE8₂₈₀₁ having a nucleic acid sequence of the coding strand whichis denoted herein as SEQ ID NO:30. Translation of SEQ ID NO:30 suggeststhat nucleic acid molecule nfE8₂₈₀, encodes a full-length flea esteraseprotein of about 59: amino acids, referred to herein as PfE8₅₉₅, havingamino acid sequence SEQ ID NO:31, assuming an open reading frame inwhich the initiation codon spans from nucleotide 99 through nucleotide101 of SEQ ID NO:30 and a stop codon spanning from nucleotide 1884through nucleotide 1886 of SEQ ID NO:30. The complement of SEQ ID NO:30is represented herein by SEQ ID NO:32. The coding region encodingPfE8₅₉₅, is represented by nucleic acid molecule nfE8₁₇₈₅, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:34and a complementary strand with nucleic acid sequence SEQ ID NO:35. Theproposed mature protein, denoted herein as PfE8₅₇₀, contains about 570amino acids which is represented herein as SEQ ID NO:55. The nucleicacid molecule encoding PfE8₅₇₀ is denoted herein as nfE8₁₇₁₀ and has acoding strand having the nucleic acid sequence SEQ ID NO:33. The aminoacid sequence of PfE8₅₉₅ (i.e., SEQ ID NO:31) predicts that PfE8₅₉₅ hasan estimated molecular weight of about 68.6 kD and an estimated pI ofabout 6.1.

Comparison of nucleic acid sequence SEQ ID NO:30 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:30 showed themost homology, i.e., about 46% identity, between SEQ ID NO:30 and a Musmusculus carboxyl ester lipase gene. Comparison of amino acid sequenceSEQ ID NO:31 (i.e., the amino acid sequence of PfE8₅₉₅) with amino acidsequences reported in GenBank indicates that SEQ ID NO:31 showed themost homology, i.e., about 28% identity between SEQ ID NO:31 andestalpha-2 esterase of Culex pipiens quinque fasciatus.

3. Nucleic acid molecule nfE3₄₂₁ was labeled with ³²P and used as aprobe to screen the prepupal cDNA library using standard hybridizationtechniques (e.g., Sambrook et al., ibid.). Two clones were isolated. Oneclone included about a 1900 nucleotide insert, referred to herein asnfE9₁₉₀₀. Nucleic acid sequence as obtained using standard techniquesfrom nfE9₁₉₀₀, to yield a flea esterase nucleic acid molecule namednfE9₂₀₀₇ having nucleic acid sequence of the coding strand which isdenoted herein as SEQ ID NO:36. Translation of SEQ ID NO:36 suggeststhat nucleic acid molecule nfE9₂₀₀₇ encodes a full-length flea esteraseprotein of about 528 amino acids, referred to herein as PfE9₅₂₈, havingamino acid sequence SEQ ID NO:37, assuming an open reading frame inwhich the initiation codon spans from nucleotide 11 through nucleotide13 of SEQ ID NO:36 and a stop co ion spanning from nucleotide 1595through nucleotide 1597 of SEQ ID NO:36. The complement of SEQ ID NO:36is represented herein by SEQ ID NO:38. The coding region encodingPfE9₅₂₈, is represented by nucleic acid molecule nfE9₁₅₈₄, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:51and a complementary strand with nucleic acid sequence SEQ ID NO:52. Theamino acid sequence of PfE9₅₂₈ (i.e., SEQ ID NO:37) predicts thatPfE9₅₂₈ has an estimated molecular weight of about 60 kD and anestimated pI of about 5.43.

Comparison of nucleic acid sequence SEQ ID NO:36 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:36 showed themost homology, i.e., about 47% identity, between SEQ ID NO:36 and ahamster mRNA for carboxylesterase precursor gene. Comparison of aminoacid sequence SEQ ID NO:37 (i.e., the amino acid sequence of PfE9₅₂₈)with amino acid sequences reported in GenBank indicates that SEQ IDNO:37 showed the most homology, i.e., about 37% identity between SEQ IDNO:37 and alpha esterase protein from Drosophila melanogaster.

As is the case for any of the nucleic acid molecules described in thisexample, variations between sequences may be due to a number of factors,such as but not limited to, sequencing errors or allelic variation.

4. Nucleic acid molecule nfE1₄₀₁ was labeled with ³²P and used as aprobe to screen the mixed instar cDNA library using standardhybridization techniques (e.g., Sambrook et al., ibid.). A clone wasisolated that included about a 2000 nucleotide insert, referred toherein as nfE10₂₀₀₀. Nucleic acid sequence as obtained using standardtechniques from nfE10₂₀₀₀, to yield a flea esterase nucleic acidmolecule named nfE10₁₉₈₇ having nucleic acid sequence of the codingstrand which is denoted herein as SEQ ID NO:67. Translation of SEQ IDNO:67 suggests that nucleic acid molecule nfE10₁₉₈₇ encodes afull-length flea esterase protein of about 530 amino acids, referred toherein as PfE10₅₃₀, having amino acid sequence SEQ ID NO:68, assuming anopen reading frame in which the initiation codon spans from nucleotide231 through nucleotide 233 of SEQ ID NO:67 and a stop codon spanningfrom nucleotide 1821 through nucleotide 1823 of SEQ ID NO:67. Thecomplement of SEQ ID NO:67 is represented herein by SEQ ID NO:69. Thecoding region encoding PfE10₅₃₀, is represented by nucleic acid moleculenfE10₁₅₉₀, having a coding strand with the nucleic acid sequencerepresented by SEQ ID NO:70 and a complementary strand with nucleic acidsequence SEQ ID NO:71. The amino acid sequence of PfE10₅₃₀ (i.e., SEQ IDNO:68) predicts that PfE10₅₃₀ has an estimated molecular weight of about59.5 kD and an estimated pI of about 5.5.

Comparison of nucleic acid sequence SEQ ID NO:67 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:67 showed themost homology, i.e., about 48% identity, between SEQ ID NO:67 and aLucilia cuprina alpha esterase gene (genemb1 #U56636) gene. Comparisonof amino acid sequence SEQ ID NO:68 (i.e., the amino acid sequence ofPfE10₅₃₀) with amino acid sequences reported in GenBank indicates thatSEQ ID NO:68 showed the most homology, i.e., about 30% identity betweenSEQ ID NO:68 and Culex pipens esterase b1 precurser protein (swissprot#P16854).

As is the case for any of the nucleic acid molecules described in thisexample, variations between sequences may be due to a number of factors,such as but not limited to, sequencing errors or allelic variation.

Example 12

This Example demonstrates the production of esterase proteins of thepresent invention in E. coli cells.

A. Flea esterase protein PHIS-PfE7₅₇₀ and flea esterase proteinPHIS-PfE8₅₇₀ were produced in the following manner. A pair of primersw,as used to amplify DNA from flea esterase nucleic acid moleculenfE7₂₈₃₆ or nfE8₂₈₀₁ produced as described in Example 11. A sense primercontaining an XhoI site (shown in bold) having the nucleic acid sequence5′ TGTGCTCGAG ATGGGATAAC CTAGATCAGC ATTTGTGC 3′ (denoted SEQ ID NO:47),was used in combination with an anti-sense primer containing a KpnI site(shown in bold) having the nucleic acid sequence 5′ TTAAGGTACCTCATCTAATA CTTCCTTCAT TACAG 3′ (denoted SEQ ID NO:48). A PCR product wasderived from nfE7₂₈₃₆, and is referred to herein as nfE7₁₇₁₀, havingnucleic acid sequence SEQ ID NO:27. The PCR product was digested withXhoI and KpnI restriction endonucleases, gel purified and subcloned intoexpression vector pTrcHisB (available from InVitrogen). The resultantrecombinant molecule, referred to herein as pTrc-nfE7₁₇₁₀, wastransformed into E. coli HB101 competent cells (available from GibcoBRL) to form recombinant cell E. coli:pTrc-nfE7₁₇₁₀.

The PCR product derived from nfE8₂₈₀₁ using the primers is referred toherein as nfE8₁₇₁₀, having nucleic acid sequence SEQ ID NO:33. PCRproduct nfE8₁₇₁₀ was digested with XhoI and KpnI restrictionendonucleases, gel purified and subcloned into expression vectorpTrcHisB. The resultant recombinant molecule, referred to herein aspTrc-nfE8₁₇₁₀, was transformed into E. coli HB101 competent cells toform recombinant cell E. coli:pTrc-nfE8₁₇₁₀.

The recombinant cells were cultured in enriched bacterial growth mediumcontaining 0.1 mg/ml ampicillin and 0.1% glucose at about 32° C. Whenthe cells reached an OD₆₀₀ of about 0.4-0.5, expression of recombinantprotein was induced by the addition of 0.5 mMisopropyl-B-D-thiogalactoside (IPTG), and the cells were cultured forabout 2 hours at about 32° C. Immunoblot analysis of recombinant cell E.coli:pTrc-nfE7₂₇₁₀ and E. coli:pTrc-nfE8₁₇₁₀ lysates using a T7 tagmonoclonal antibody (available from Novagen, Inc., Madison, Wis.)directed against the fusion portion of the recombinant PHIS-PfE7₅₇₀ andPHIS-PfE8₅₇₀ fusion proteins identified proteins of appropriate size,namely an about 65 kD protein for each fusion protein.

B. Flea esterase protein PHIS-PfE6₅₄₀ was produced in the followingmanner. A pair of primers was used to amplify DNA from flea esterasenucleic acid molecule nfE6₁₇₉₂ produced as described in Example 11. Asense primer containing an XhoI site having the nucleic acid sequence 5′AAACTCGAGT CCCCCGACTG TAACTTTGC 3′ (denoted SEQ ID NO:62; XhoI siteshown in bold), was used in combination with an anti-sense primercontaining a PstI site having the nucleic acid sequence 5′ TCATCTGCAGTTATTGACTG TGCAAAGTTT TTGTGG 3′ (denoted SEQ ID NO:63; PstI site shownin bold). A PCR product was derived from nfE6₁₇₉₂, and is referred toherein as nfE6₁₄₈₈, having nucleic acid sequence SEQ ID NO:76. The PCRproduct was Digested with XhoI and PstI restriction endonucleases, gelpurified and subcloned into expression vectorlambdaP_(R)/T²ori/S10HIS-RSET-A9, that had been digested with XhoI andPstI and dephosphorylated. The resultant recombinant molecule, referredto herein as pCro-nfE6₁₄₈₈, was transformed into E. coli HB101 competentcells (available from Gibco BRL) to form recombinant cell E.coli:pCro-nfE6₁₄₈₈.

The recombinant cells were cultured using the method generally describedin Section A of this example, except that the cells were grown underheat shift conditions rather than in the presence of IPTG. The cellswere grown at 32° C. for about 2 hours, and then grown at 42° C.Immunoblot analysis of recombinant cell E. coli:pCro-nfE6₁₄₈₈ lysateusing a T7 tag monoclonal antibody directed against the fusion portionof the recombinant PHIS-PfE6₅₄₀ fusion protein identified proteins ofappropriate size, namely an about 60 kD protein for each fusion protein.

Expression of the recombinant PHIS-PfE6₅₄₀ fusion protein was improvedby transforming supercoiled plasmid pCro-nfE6₁₄₈₈ DNA harvested from E.coli:pCro-nfE6₁₄₈₈ cells into the BL-21 strain of E. coli (availablefrom Novagen). The amount of expression PHIS-PfE6₅₄₀ was confined byimmunoblot using the method described immediately above.

E. coli cells expressing PHIS-PfE6₅₄₀ protein were harvested from about2 liters of media and suspended in about 140 ml of 50 mM Tris, pH 8.0,50 mM NaCl, 0.1 mM phenylmethylsulfonylfluoride (PMSF) (SolubilizationBuffer). The cells were broken by passage through a microfluidizer at 30psi for 30 cycles. Tile sample was centrifuged at about 16,000×g for 30mile at 4° C. The supernatant (S1) was recovered and the pellet wasresuspended in about 80 ml of Solubilization Buffer and centrifuged atabout 16,000×g for 30 min at 4° C. The supernatant (S2) was recoveredand the pellet was resuspended in about 80 ml of Solubilization Buffercontaining 0.1% Triton-X100 and centrifuged at about 16,000×g for 30 minat 4° C. The supematant (S3) was recovered and the pellet wasresuspended in about 140 mls 50 mM Tris, pH 8.0, 8 M Urea, 0.1 M PMSFand centrifuged at about 16,000×g. The supernatant (S4) was recoveredand the pellet was resuspended in 40 mls 50 mM Tris, 8 M Urea, 0.1 MPMSF. Aliquots of each pellet and supernatant were anally, by SDS-PAGEand immunoblot using the T7 tag monoclonal antibody described above. Theresults indicated that the PHIS-PfE6₅₄₀ protein was located in the finalsupematant (S4). The PHIS-PfE6₅₄₀ protein was loaded onto a 5.0 ml,Metal chelating HiTrap column charged with NiCl₂ (obtained fromPharmacia Biotech Inc., Piscataway, N.J.), previously equilibrated with50 mM Tris, 1 mM PMSF, 1 mM β-mercaptoethanol (βME), 8 M urea, pH 8.0(Buffer A). The column was washed with 10 column volumes (cv) of BufferA and then with 10 cv with 50 mM Tris, 25 mM sodium acetate, 1 mM PMSF,1 mM βME, 8 M urea, pH 6.0 (Buffer B) to remove loosely bound proteins.Bound PHIS-PfE6₅₄₀ protein was eluted with 10 cv of 50 mM Tris, 25 mMsodium acetate, 1 mM PMSF, 1 mM βME, 8 M urea, pH 4.0 (Buffer C). Columnfractions were analyzed for the presence of PHIS-PfE6₅₄₀ protein byimmunoblot using the T7 tag monoclonal antibody as described above. Theresults indicated that the majority of the PHIS-PfE6₅₄₀ protein waseluted by Buffer C. The fractions containing the PHIS-PfE6₅₄₀ proteinwere combined and loaded onto a 5 ml SP-Sepharose HiTrap column(obtained from Pharmacia Biotech Inc.) previously equilibrated with 50mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM βME, 8 M Urea, pH 4.5(SP-Sepharose Buffer). The column was washed with SP-Sepharose Bufferuntil most of the unbound protein was removed. Bound protein was elutedwith an increasing salt gradient to 1 M NaCl over 100 ml (20 cv) inSP-sepharose buffer. Column fractions were analyzed for the presence ofPHIS-PfE6₅₄₀, protein by immunoblot using the T7 tag monoclonal antibodyas described above. The results indicated that the PHIS-PfE6₅₄₀ proteinwas eluted at about 0.75 M NaCl.

The purified PHIS-PfE6₄₈₈ protein was used to produce an anti-M6polyclonal antiserum as follows. Rabbits were immunized withPHIS-PfE6₁₄₈₈ protein diluted to a concentration of about 0.1 mg/ml inPBS. One milliliter of the dilution was mixed 1:1 mix with CompleteFreunds Adjuvant. In the primary immunization, about 500 μl of the 1:1mix was injected subcutaneously into 5 different sites (0.1 ml/site) and500 μl was injected intradermally into 5 different sites (0.1 ml/site)on the rabbit. Booster shots were administered to the rabbitintramuscularly in 4 sites using 250 μl/site of a 1:1 mix ofPHIS-PfE6₁₄₈₈ protein with Incomplete Freunds Adjuvant. The boostershots were administered at days 14 and 35. Serum samples were obtainedprior to immunization (pre-bleed), and at day 14 after primaryimmunization and day 14 after the first and second boost.

C. Flea esterase protein PHIS-PfE9₅₂₈ was produced in the followingmanner. A pair of primers was used to amplify DNA from flea esterasenucleic acid molecule nfE9₂₀₀₇ produced as described in Example 11. Asense primer containing an BamHI site having the nucleic acid sequence5′-TTC CGG ATC CGG CTG ATC TAC AAG TGA CTT TG-3′ (denoted SEQ ID NO:64;BamHI site shown in bold), was used in combination with an anti-senseprimer containing a XhoI site having the nucleic acid sequence 5′ TGGTAC TCG AGT CAT AAA AAT TTA TTC CAA AAT C 3′ (denoted SEQ ID NO:65; XhoIsite shown in bold). A PCR product was derived from nfE9₂₀₀₇, and isreferred to herein as nfE9₁₅₄₀, having nucleic acid sequence SEQ IDNO:51. The PCR product was digested with BamHI and XhoI restrictionendonucleases, gel purified and subcloned into expression vectorpTrcHisB (available from InVitrogen). The resultant recombinantmolecule, referred to herein as pTrc-nfE9₅₁₄₀, was transformed into E.coli HB101 competent cells (available from Gibco BRL) to formrecombinant cell E. coli:pTrc-nfE9₁₅₄₀.

The recombinant cells were cultured using the method described inSection A of this example. Immunoblot analysis of recombinant cell E.coli:pTrc-nfE9₁₅₄₀ lysate using a T7 tag monoclonal antibody directedagainst the fusion portion of the recombinant PHIS-PfE9₅₂₈ fusionprotein identified proteins of appropriate size, namely an about 59 kDprotein for each fusion protein.

Expression of the recombinant PHIS-PfE9₅₂₈ fusion protein was improvedby transforming supercoiled plasmid pTrc-nfE9₁₅₈₄ DNA harvested from E.coli:pTrc-nfE9₁₅₄₀ cells into the BL-21 strain of E. coli. The amount ofexpression PHIS-PfE9₅₂₈ was confined by immunoblot using the methoddescribed immediately above.

Two liters of media from cultures of E. coli cells expressingPHIS-PfE9₅₂₈ protein were harvested and S4 supematant was prepared usingthe method described above in section B. The PHIS-PfE9₅₂₈ proteincontained in the S4 supernatant was loaded onto a 5.0 ml, Metalchelating HiTrap column charged with NiCl₂ (available from PharmaciaBiotech Inc., Piscataway, N.J.), previously equilibrated with 50 mMTris, 1 mM PMSF, 1 mM βME, 8 M urea, pH 8.0 (Buffer A). The column waswashed with 5 cv of Buffer A until all unbound protein was removed.Bound protein was eluted with a linear gradient from Buffer A to 50 mMTris, 1 mM PMSF, 1 mM βME, 8 M urea, 1 M NaCl, pH 4.0. Column fractionswere analyzed for the presence of PHIS-PfE9₅₂₈ protein by immunoblotusing the T7 tag monoclonal antibody as described above. The resultsindicated that the majority of the PHIS-PfE9₅₂₉ protein was eluted atabout 250 mM NaCl. The fractions containing the PHIS-PfE9₅₂₈ proteinwere combined and loaded onto a C4-reversed phase column (obtained fromVydak, Hesperia, Calif.), previously equilibrated with 0.05%trifluoroacetic acid (TFA). The column was washed with 0.05% TFA untilall unbound protein was removed. Bound proteins were eluted with alinear gradient from 0.05% TFA to 0.05% TFA in acetonitrile. Columnfractions were analyzed for the presence of PHIS-PfE9₅₂₈ protein byimmunoblot using the T7 tag monoclonal antibody as described above. Theresults indicated that the PHIS-PfE9₅₂₈ protein was eluted at about 40%acetonitrile. The fractions containing the PHIS-PfE9₅₂₈ protein werecombined and loaded onto a 5 ml Q-Sepharose HiTrap column previouslyequilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM βME,8 M Urea, pH 8.5 (Q-Sepharose Buffer). The column was washed withQ-Sepharose Buffer until all unbound protein was removed. Bound proteinwas eluted with an increasing salt gradient to 1 M NaCl over 100 ml (20cv) in Q-sepharose buffer. Column fractions were analyzed for thepresence of PHIS-PfE9₅₂₈ protein by immunoblot using the T7 tagnoncolonial antibody as described above. The results indicated that thePHIS-PfE9₅₂₈ protein was eluted at about 0.3 M NaCl.

The purified PHIS-PfE9₅₂₈ protein was used to produce an anti-P1polyclonal antiserum as follows. Rabbits were immunized withPHIS-PfE9₅₂₈ protein diluted to a concentration of about 0.1 mg/ml inPBS. One milliliter of the dilution was mixed 1:1 mix with CompleteFreunds Adjuvant. In the primary immunization, about 500 μl of the 1:1mix was injected subcutaneously into 5 different sites (0.1 ml/site) and500 μl was injected intradermally into 5 different sites (0.1 ml/site)on the rabbit. Booster shots were administered to the rabbitintramuscularly in 4 sites using 250 μl/site of a 1:1 mix ofPHIS-PfE9₅₂₈ protein with Incomplete Freunds Adjuvant The booster shotswere administered at days 14 and 35. Serum samples were obtained priorto immunization (pre-bleed), and at day 14 after primary immunizationand day 14 after the first and second boost.

D. Flea esterase protein PHIS-PfE7₂₇₅ was produced in the followingmanner. A 650-bp fragment was produced by digesting nfE7₂₈₃₆ DNA withthe restriction enzymes BamHI and BglII. The BamHI and BglII fragmentderived from nfE7₂₈₃₆ is referred to herein as nfE7₆₅₀, having nucleicacid sequence SEQ ID NO:72 and amino acid SEQ ID NO:73. The fragment waspurified using a Qiaquick™ Kit (available from Qiagen, Santa Clarita,Calif.), according to methods provided by the manufacturer. The purifiedfragment was subcloned into expression vector pTrcHisC which had beendigested with BamHI and BglII. The resultant recombinant molecule,referred to herein as pTrc-nfE7₆₅₀ was transformed into E. coli DH-5acompetent cells (available from Gibco BRL) to form recombinant cell E.coli:pTrc-nfE7₆₅₀.

The recombinant cells were cultured using the method described above insection A. Immunoblot analysis of recombinant cell E. coli:pTrc-nfE7₆₅₀Pulsate using a T7 tag monoclonal antibody directed against the fusionportion of the recombinant PHIS-PfE7₂₇₅ fusion protein identifiedproteins of appropriate size, namely an about 35 kD protein for eachfusion protein.

Expression of the recombinant fusion protein was improved bytransforming supercoiled plasmid pTrc-nfE7₆₅₀ DNA harvested from E.coli:pTrc-nfE7₆₅₀ cells into the BL-21 strain of E. coli. The amount ofexpression E. coli:pTrc-nfE7₆₅₀ was confined by immunoblot using themethod described immediately above.

Example 13

This Example demonstrates the production of esterase proteins of thepresent invention in eukaryotic cells.

A. Recombinant molecule pBv-nfE7₁₇₈₈, containing a flea esterase nucleicacid molecule spanning nucleotides from about 99 through about 1886 ofSEQ ID NO:24, and pBv-nfE⁸ ₁₇₈₈, containing a flea esterase nucleic acidmolecule spanning nucleotides from about 99 through about 1883 of SEQ IDNO:30 each, operatively linked to baculovirus polyhedron transcriptioncontrol sequences were produced in the following manner. In order tosubclone a flea esterase nucleic acid molecule into baculovirusexpression vectors, flea esterase nucleic acid molecule-containingfragments were separately PCR amplified from nfE7₂₈₃₆ or nfE8₂₈₀₁ DNA. APCR fragment of 1858 nucleotides, named nfE7₁₈₅₈, was amplified fromnfE7₂₈₃₆ using a sense primer E1113 FWD having the nucleic acid sequence5′-AAAACTGCAG TATAAATATG TTACCTCACA GTAGTG-3′ (SEQ ID NO:49; PstI siteshown in bold) and an antisense primer E1113/2212 REV having the nucleicacid sequence 5′-TGCTCTAGAT TATCTAATAC TTCCTTCATT ACAG (SEQ ID NO:50;XbaI site shown in bold). A PCR fragment of 1858 nucleotides, namednfE8₁₈₅₈, was amplified from nfE8₂₈₀₁ using a sense primer E2212 FWDhaving the nucleic acid sequence 5′-AAACTGCAG TATAAATATG TTACCTCACAGTGCATTAG-3′ (SEQ ID NO:66; PstI site shown in bold), and the antisenseprimer E1113/2212 REV. The N-terminal primer was designed from the pol hsequence of baculovirus with modifications to enhance expression in thebaculovirus system.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PfE7₅₉₆, the about 1,802 base pair PCRproduct (referred to as Bv-nfE7₁₈₀₂) was digested with PstI and XbaI andsubcloned into unique PstI and XbaI sites of pVL1392 baculovirus shuttleplasmid (available from Pharmingen, San Diego, Calif.) to produce therecombinant molecule referred to herein as pVL-nfE7₁₈₀₂.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PfE8₅₉₅, the about 1,792 base pair PCRproduct (referred to as Bv-nfE8₁₇₉₂) w,as digested with PstI and XbaIand subcloned into PstI and XbaI digested to produce the recombinantmolecule referred to herein as pVL-nfE8₁₇₉₂.

The resultant recombinant molecules, pVL-nfE7₁₈₀₂ and pVL-nfE8₁₇₉₂, wereverified for proper insert orientation by restriction mapping. Such arecombinant molecule can be co-transfected with a linear Baculogoldbaculovirus DNA (available from Pharmingen) into S. frugiperda Sf9 cells(available from InVitrogen) to form the recombinant cells denoted S.frugiperda:pVL-nfE7₁₈₀₂ and S. frugiperda:pVL-nfE8₁₇₉₂ . S.frugiperda:pVL-nfE7₁₈₀₂ can be cultured in order to produce a fleaesterase protein PfE7₅₉₆ . S. frugiperda:pVL-nfE8₁₇₉₂ can be cultured inorder to produce a flea esterase protein PfE8₅₉₅.

B. Recombinant molecule pBv-PfE9₅₂₈, containing a flea esterase nucleicacid molecule spanning nucleotides from 14 through 1595 of SEQ ID NO:36,operatively linked to baculovirus polyhedron transcription controlsequences were produced in the following manner. In order to subclone aflea esterase nucleic acid molecule into baculovirus expression vectors,a flea esterase nucleic acid molecule-containing fragment was PCRamplified from nfE9₂₀₀₇ DNA. A PCR fragment of about 1600 nucleotides,named nfE9₁₆₀₀, was amplified from nfE9₂₀₀₇ using a sense primer P121B1Sense having the nucleic acid sequence 5′-CGC GGA TCC GCT GAT CTA CAAGTG ACT TTG C-3′ (SEQ ID NO:75; BamHI site shown in bold) and anantisense primer PI21B1 Anti having the nucleic acid sequence 5′-CCG AGCGGC CGC ATA AAA ATT TAT TCC AAA ATC TAA GTC G-3′ (SEQ ID NO:76; NotIsite shown in bold). The N-terminal primer was designed from the pol hsequence of baculovirus with modifications to enhance expression in thebaculovirus system.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PfE9₅₂₈, the about 1,600 base pair PCRproduct (referred to as Bv-nfE9₁₆₀₀) was digested with BamHI and NotIand subcloned into unique BamHI and NotI sites of pVL3193 baculovirusshuttle plasmid (available from Pharmingen, San Diego, Calif.) toproduce the recombinant molecule referred to herein as pVL-nfE9₁₆₀₀.

The resultant recombinant molecule, pVL-nfE9₁₆₀₀, was verified forproper insert orientation by restriction mapping. Such a recombinantmolecule can be co-transfected with a linear Baculogold baculovirus DNAinto S. frugiperda Sf9 cells to form the recombinant cells denoted S.frugiperda:pVL-nfE9₁₆₀₀ . S. frugiperda:pVL-nfE9₁₆₀₀ can be cultured inorder to produce a flea esterase protein PfE9₅₂₉.

An immunoblot of supernatant from cultures of S. frugiperda:pL-nfE9₁₆₀₀cells producing the flea esterase protein PfE9₅₂₈ was performed usingthe anti-P1 polyclonal antiserum described in detail in Example 12.Blots were incubated using serum samples from the pre-bleed or fromserum collected 14 days after the first boost of the rabbit. Analysis ofthe supernatent from cultures of S. frugiperda:pVL-nfE9₁₆₀₀ cellsidentified an about 66 kD protein

C. Recombinant molecule pBv-PfE6₅₃₀, containing a flea esterase nucleicacid molecule spanning nucleotides from 50 through 1701 of SEQ ID NO:18,operatively linked to baculovirus polyhedron transcription controlsequences ere produced in the following manner. In order to subclone aflea esterase nucleic acid molecule into baculovirus expression vectors,a flea esterase nucleic acid molecule-containing fragment was PCRamplified from nfE6₁₇₉₂ DNA. A PCR fragment of about 1679 nucleotides,named nfE10₁₆₇₉, was amplified from nfE6₁₇₉₂ using a sense primer M6M32Sense having the nucleic acid sequence 5′-GCG AGG CCT TAT AAA TAT GTCTCG TGT TAT TTT TTT AAG TTG-3′ (SEQ ID NO:75; StuI site shown in bold)and an antisense primer M6M32 Anti having the nucleic acid sequence5′-GCA CTG CAG TTA TTG ACT GTG CAA AGT TTT TGT GG-3′ (SEQ ID NO:76; PstIsite shown in bold). The N-terminal primer was designed from the pol hsequence of baculovirus with modifications to enhance expression in thebaculovirus system.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PfE6₅₃₀, the about 1,679 base pair PCRproduct (referred to as Bv-nfE6₁₆₇₉) was digested with StuI and PstI andsubcloned into unique StuI and PstI sites of FAST BAC™ baculovirusshuttle plasmid (obtained from Gibco-BRL) to produce the recombinantmolecule referred to herein as pFB-nfE6₁₆₇₉.

The resultant recombinant molecule, pFB-nfE6₁₆₇₉, was verified forproper insert orientation by restriction mapping. Such a recombinantmolecule can be transformed into E. coli strain DH10 (obtained fromGibco-BRL) according to the manufacturer's instructions. ThepFB-nfE6₁₆₇₉ isolated from the transformed DH10 cells can then beco-transfected with a linear Baculogold baculovirus DNA into S.frugiperda Sf9 cells to form the recombinant cells denoted S.frugiperda:pFB-nfE6₁₆₇₉ . S. frugiperda:pFB-nfE6₁₆₇₉ can be cultured inorder to produce a flea esterase protein PfE6₅₃₀.

An immunoblot of supernatant from cultures of S. frugiperda:pFB-nf6₁₆₇₉cells producing the flea esterase protein PfE6₅₃₀ was performed usingthe anti-M6 polyclonal antiserum described in detail in Example 12.Blots were incubated using serum samples from the pre-bleed or fromserum collected 14 days after the first boost of the rabbit. Analysis ofthe supematant from cultures of S. frugiperda:pFB-nfE6₁₆₇₉ cellsidentified an about 66 kD protein.

N-terminal amino acid sequence was obtained using standard methods forthe about 66 kD protein identified using the anti-M6 polyclonalantiserum. The N-terminal amino acid sequence was determined to beidentical to the N-terminal amino acid sequence of SEQ ID NO:44.

Example 14

This example describes the purification of carboxylesterase protein fromfed flea midguts.

About 43,000 cat blood-fed adult flea midguts were collected andprepared as previously described in Example 1. The extract was thenadded in 2 aliquots to columns containing about 1 to about 2 ml ofp-aminobenzamidine linked agarose beads (available from Sigma),equilibrated in 50 mM Tris (pH 8.0), 400 mM NaCl, and incubatedovernight at 4° C. The columns were then drained to remove unboundprotein and the two aliquots of unbound protein were combined. Thecollected unbound protein was then concentrated and diafiltered into atotal volume of about 16 ml of 25 mM Tris (pH 8), 10 mM NaCl using anUltrafree-20 10 kD centrifugal concentrator (available from Millipore;Bedford, Mass.).

Aliquots of about 8 ml were loaded onto an Uno Q6 anion exchange column(available from Bio-Rad, Hercules, Calif.) equilibrated in 25 mM Tris(pH 8), 10 mM NaCl, operated on a BioLogic liquid chromatography system(available from Bio-Rad). The column was washed with 25 mM Tris (pH 8),10 mM NaCl until all unbound protein was removed. Protein bound to thecolumn was then eluted with a linear gradient from 10 mM to 1 M NaCl in25 mM Tris, pH 8. Fractions were assayed for CE activity using the assaydescribed previously. The results indicated that CE activity was elutedat about 220 mM NaCl.

Fractions containing CE activity were pooled and diafiltered into atotal volume of about 3 ml of 20 mM MES buffer(2-N-morpholino)ethanesulfonic acid), pH 6.0, containing 10 mM NaCl, inpreparation for cation exchange chromatography. The sample was thenapplied to an Uno S1 cation exchange column (available from Bio-Rad)equilibrated in MES buffer. The column was washed with MES buffer untilall unbound protein was removed. Protein bound to the column was theneluted with a linear gradient from 10 mM to 1 M, NaCl in 20 mM MESbuffer, pH 6. Fractions were assayed for CE activity using the assaydescribed previously. Tile results indicated that CE activity was notretained on the cation exchange column using the above conditions, andall of the activity was found in the flow-through fractions.

Fractions containing CE activity were pooled and diafiltered into atotal volume of about 3 ml of 25 mM Tris (pH 8), 10 mM NaCl, inpreparation for an additional anion exchange chromatography step. Thesample was then applied to a Bio-Scale Q2 anion exchange column(available from Bio-Rad). The column was washed with 25 mM Tris (pH 8),10 mM NaCl until all unbound protein was removed. Protein bound to thecolumn was then eluted with a linear gradient from 10 mM to 1 M NaCl in25 mM Tris, pH 8. Fractions were assayed for CE activity using the assaydescribed previously. The results indicated that CE activity was elutedat about 130 mM NaCl.

A fraction containing CE activity was diluted into a total volume ofabout 4 ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl, inpreparation for hydroxyapatite chromatography. The sample was thenapplied to a Bio-Scale CHT2-I column (available from Bio-Rad) at a flowrate of about 0.5 ml/min. The column was washed with 10 mM phosphatebuffer, pH 7.2 containing 10 mM NaCl until all unbound protein wasremoved. Protein bound to the column was then eluted with a lineargradient from 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl to0.5 M 10 mM phosphate buffer, pH 6.5 containing 10 mM NaCl. Fractionswere assayed for CE activity using the assay described previously. Theresults indicated that CE activity as eluted at about 200 mM phosphate.

Example 15

This example describes the purification of a carboxylesterase proteinfrom wandering flea larvae.

About 120,000 bovine blood-fed adult wandering flea larvae werehomogenized in 3 batches of about 40,000 wandering larvae in each batch,in Tris buffered saline (TBS), pH 8.0 as previously described, exceptthat about 1.2 mg of phenylthiourea was added to each ml of TBS duringthe extraction procedure to inhibit cross linkin, reactions. Theextracts were dialyzed against 2 changes of about 2 L of 10 mM phosphatebuffer, pH 7.2 containing 10 mM NaCl in preparation for hydroxyapatitebatch chromatography. The samples were then filtered through glassAcrodiscs® (available from Gelman Sciences, Ann Arbor, Mich.) and addedto 14 g of Macro-Prep Ceramic Hydroxyapatite, Type I, 40 μm beads(available from Bio-Rad), previously equilibrated in 10 mM phosphatebuffer, pH 7.2 containing 10 mM NaCl. The extracts and beads were rockedat room temperature for about 30 minutes. Following incubation, thebeads were centrifuged for about 5 minutes at 500×g and the supernatantsremoved. The beads were washed with about 40 ml 10 mM phosphate buffer,pH 7.2 containing 10 mM NaCl, centrifuged as above, and washed andcentrifuged again to eliminate all unbound protein. Bound proteins wereeluted by washing the beads with about 40 ml of each of 100 mM, 200 mM,300 mM, and 400 mM phosphate buffer, pH 6.5 containing 10 mM NaCl.Following elution, the supernatants from each concentration of phosphatebuffer were tested for juvenile hormone esterase activity as describedpreviously in Example 7. The juvenile hormone esterase activity elutedat different phosphate concentrations in each batch, but the activitywas generally found in the 200 mM to 300 mM phosphate fractions.

The fractions that contained the highest juvenile hormone esteraseactivity were combined and diafiltered into a total volume of about 50ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl using astirred cell concentrator fitted with a YM10 ultrafiltration membrane(available from Anicon, Beverly, Mass.). Aliquots of about 5 ml to 10 mlwere applied to a chromatography column containing about 10 ml ofMacro-Prep Ceramic Hydroxyapatite, Type I, 20 μm beads, previouslyequilibrated with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl.The column was washed with 10 mM phosphate buffer, pH 7.2 containing 10mM NaCl until all unbound protein as removed. Protein bound to thecolumn was then eluted with a linear gradient from 10 mM phosphatebuffer, pH 7.2 containing 10 mM NaCl to 0.5 M 10 mM phosphate buffer, pH6.5 containing 10 mM NaCl. Fractions were assayed for carboxylesteraseactivity using the assay described previously. The results indicatedthat carboxylesterase activity was eluted at about 160 mM phosphate.

The fractions that contained the highest carboxylesterase activity werecombined and diafiltered into a total volume of about 15 ml of 20 mMsodium acetate buffer, pH 4.0 in preparation for cation exchangechromatography. Aliquots of about 3 ml were applied to a PolyCat Acation exchange column (available from PolyLC, Columbia, Md.)equilibrated in 20 mM sodium acetate buffer, pH 6.0, operated on aWaters high performance liquid chromatography system (available fromWaters Corporation, Milford, Mass.). The column was washed with 20 mMsodium acetate buffer, pH 6.0 until all unbound protein was removed.Protein bound to the column was then eluted with a linear gradient from20 mM sodium acetate buffer, pH 6.0 to 20 mM sodium acetate buffer, pH6.0 containing 1 M NaCl. Fractions were assayed for CE activity usingthe assay described previously. The results indicated that there weretwo pools of CE activity. The first pool was not retained on the cationexchange column, and the second pool was eluted at about 170 mM NaCl.

The fractions from the second pool that contained the highestcarboxylesterase activity were combined and diafiltered into a totalvolume of about 10 ml of 25 mM Tris (pH 8), 10 mM NaCl, in preparationfor anion exchange chromatography. The sample was then applied to aBio-Scale Q2 anion exchange column (available from Bio-Rad). The columnwas washed with 25 mM Tris (pH 8), 10 mM NaCl until all unbound proteinwas removed. Protein bound to the column was then eluted with a lineargradient from 10 mM to 1 M NaCl in 25 mM Tris, pH 8. Fractions wereassayed for carboxylesterase activity using the assay describedpreviously. The results indicated that carboxylesterase activity waseluted at about 350 mM NaCl.

Fractions containing carboxylesterase activity were combined andconcentrated to about 175 μl using a Centricon 10 centrifugalconcentrator (available from Amicon, Beverly, Mass.) in preparation forsize exclusion chromatography. The sample was applied to a Bio-SelectSEC 125-5 size exclusion chromatography column (available from Bio-Rad),previously equilibrated in TBS, pH 7.2. About 250 μl fractions were thencollected. The fractions were assayed for carboxylesterase activityusing the assay described previously. The results indicated that thecarboxylesterase activity was eluted in about 5.5 to 6 ml of buffer,corresponding to a molecular weight of about 40 to 100 kDa based on theelution volumes of gel filtration molecular weight standard proteins(available from Sigma, St. Louis, Mo.).

Example 16

This example describes the purification of juvenile hormone esteraseactivity from unfed adult flea midguts by affinity chromatography.

About 16,000 unfed adult flea midguts were collected in 20 mM Trisbuffer (pH 7.7), containing 130 mM NaCl, 1 mM sodium EDTA, 1 mMPefabloc® (available from Boehringer Mannheim, Indianapolis, Ind.), 1microgram/ml (μg/ml) leupeptin and 1 μg/ml pepstatin. The midguts werehomogenized by freeze-fracture and sonication, and then centrifuged atabout 14,000×g for 20 min. The soluble material from the centrifugationstep was recovered, diafiltered into Tris buffered saline (TBS), andapplied to a disposable plastic column containing about 1 ml of3-[(4′-mercapto)butylthio]-1,1,1-trifluoropropan-2-one linked Sepharose6B beads, prepared similarly to the method described by Venkatesh et al.(J. Biol. Chem., Vol. 265, No. 35, 21727-21732, 1990) (the3-[(4′-mercapto)butylthio]-1,1,1-trifluoropropan-2-one was a gift fromNovartis Corp., Basel, Switzerland; and the Epoxy-activated Sepharose 6Bis available from Pharmacia Biotech Inc., Piscataway, N.J.). Afterovernight incubation at 4° C., the column was drained and the beads werewashed with about 10 ml TBS, then about 10 ml TBS containing 0. 1% (w/v)n-octylglucoside (OG; available from Boehringer Mannheim). Thepre-column, flow-through, and wash fractions were tested for juvenilehormone esterase activity by the method previously described above inExample 7. The results indicate that the flow-through fraction containedapproximately 40% less juvenile hormone esterase activity than thepre-column material, and that the washes contained very little activity.

Bound protein was eluted from the beads by adding about 10 ml of TBScontaining 0.1% (w/v) OG and 1 mM3-octylthio-1,1,1-trifluoropropan-2-one (OTFP; a gift from NovartisCorp.). After a 2 hour incubation at 4° C., about 5 ml of the eluate wascollected, and the remaining 5 ml was incubated with the beads overnightat 4° C. The following day, the beads were drained, the eluatecollected, and an additional 10 ml of TBS containing 0.1% (w/v) OG and 1mM OTFP was added to the beads. After an overnight incubation at 4° C.,the beads were drained and the eluate collected. Tile final 10 mlelution step was repeated 3 additional times so that we had 6 elutedfractions. The first elution fraction was dialyzed overnight twiceagainst 1 liter of fresh TBS to remove excess OTFP. The second elutionfraction was also dialyzed overnight against 1 liter of fresh TBS toremove OTFP. The third through sixth elution fractions were notdialyzed. All six eluted fractions were tested for juvenile hormoneesterase activity by the method previously described above in Example 7.The results indicate that only the third elution fraction containeddetectable juvenile hormone esterase activity. Analysis of the elutedfractions by silver-stained SDS-PAGE indicated that several proteinswere specifically bound to the affinity beads and were eluted by OTFP.The apparent molecular weights of these proteins, as determined bySDS-PAGE, were about 66 kDa, 55 kDa, and 33 kDa. About 3.5 ml of eachelution fraction were combined and concentrated to about 110 μl using aCentriplus 10 centrifugal concentrator (available from Amicon, Beverly,Mass.). This pool was separated by SDS-PAGE and blotted onto apolyvinylidene difluoride (PVDF) membrane as described previously inExample 5. The stained protein band at about 66 kDa was excised andsubjected to N-terminal sequence analysis as described previously.

The results indicated that the N-terminal amino acid sequence of theputative 66 kDa juvenile hormone esterase protein was DL y/g V k/y/cgv/q/n LQGTLKGKE (denoted herein as SEQ ID NO:74), in which the lowercase letters designate uncertainties. Below is shown a comparisonbetween different esterase amino acid sequences of the presentinvention.

SEQ ID NO:74: DL (y/g) V (k/y/g) (v/q/n) LQGTLKGKE

SEQ ID NO:37: DL Q V T L LQGTLKGKE

(Residues 3-17)

Example 17

This example describes the purification of an active recombinantjuvenile hormone esterase protein from baculovirus supematants.

About 1 liter of supernatant from cultures of S. frugiperda:pVL-nfE9₁₆₀₀cells producing the flea esterase protein PfE9₅₂₈ was brought to about50% saturation with ammonium sulfate and centrifuged at about 20000×gfor about 30 minutes at 4° C. to pellet the precipitated material. Aftercentrifugation, the pellet was retained and the supernatant was broughtto about 100% saturation with ammonium sulfate and centrifuged as above.The material in both pellets were resuspended separately in about 35 mlof Tris buffered saline (TBS), pH 8.0. The resuspended pellets wereassayed for the presence of flea esterase protein PfE9₅₂₈ using standardWestern blot techniques and a polyclonal antiserum that bindsspecifically to PfE9₅₂₈ protein. Briefly, a rabbit was immunized withPHIS-PfE9₅₂₈ protein purified from E. coli:pTrc-nfE9₁₅₈₄ cells(described above in Example 12C) and boosted using standard procedures.The results indicated that the flea esterase protein PfE9₅₂₈ was presentin the S. frugiperda:pVL-nfE9₁₆₀₀ supernatants and the protein wasprecipitated by adjusting the ammonium sulfate concentration from about50% saturation to about 100% saturation.

The resuspended flea protein PfE9₅₂₈ was diafiltered into about 10 ml of25 mM Tris (pH 8.0), 10 mM NaCl using an Ultrafree-20 10 kD centrifugalconcentrator in preparation for anion exchange chromatography. Aliquotsof about 5 ml were loaded onto an Uno Q6 anion exchange columnequilibrated in 25 mM Tris (pH 8.0), 10 mM NaCl. The column was washedwith 25 mM Tris (pH 8.0), 10 mM NaCl until most of the unbound proteinwas removed. Protein bound to the column was then eluted with a lineargradient from 10 mM to 1 M NaCl in 25 mM Tris buffer (pH 8.0). Fractionswere assayed for the presence of flea esterase protein PfE9₅₂₈ by theimmunoblot method described above. The results indicated that the fleaesterase protein PfE9₅₂₈ was eluted at about 200 mM NaCl.

Fractions containing the flea esterase protein PfE9₅₂₃ were pooled andconcentrated to about 440 μl using a Centricon 10 kD centrifugalconcentrator in preparation for size exclusion chromatography. Thesample was applied in 3 aliquots to a Bio-Select SEC 125-5 sizeexclusion chromatography column (available from Bio-Rad), previouslyequilibrated in TBS, pH 7.2. The column was eluted with TBS, pH 7.2 at aflow rate of about 0.5 ml/min, and fractions of about 250 μl werecollected. The fractions were assayed for the presence of flea esteraseprotein PfE9₅₂₈ by the immunoblot method described above. The resultsindicate ‘t° at the flea esterase protein PfE9₅₂₈ was eluted with about6 ml of buffer, corresponding to a molecular weight of about 40 to 100kDa based on the elution volumes of gel filtration molecular weightstandard proteins (available from Sigma, St. Louis, Mo.).

Fractions containing flea esterase protein PfE9₅₂₈ were then assayed forjuvenile hormone esterase activity as described in Example 7 andcarboxylesterase activity as described in Example 2. The resultsindicated that the purified flea esterase protein PfE9₅₂₈ had bothjuvenile hormone esterase activity and carboxylesterase activity.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

76 1 401 DNA Ctenocephalides felis CDS (92)..(400) 1 tttacatcattaataaacat aaatctaata aatcttgtgg atcaagatca agtttattag 60 tgagagtgttggatttgtga aatatttcaa a atg aat tct tta att gta aaa 112 Met Asn Ser LeuIle Val Lys 1 5 att tct caa gga gct att gag ggg aag gaa atg att aat gataat gga 160 Ile Ser Gln Gly Ala Ile Glu Gly Lys Glu Met Ile Asn Asp AsnGly 10 15 20 aag tcg ttt aga gga ttt ttg ggt ata cct tat gct aaa ccg cctata 208 Lys Ser Phe Arg Gly Phe Leu Gly Ile Pro Tyr Ala Lys Pro Pro Ile25 30 35 gga aat ctt ana ttt aag cct cct caa aag cct gat gat tgg aat gat256 Gly Asn Leu Xaa Phe Lys Pro Pro Gln Lys Pro Asp Asp Trp Asn Asp 4045 50 55 gtt cga cca gct act gaa naa gca aat ggt tgt aga tcg aaa cat atg304 Val Arg Pro Ala Thr Glu Xaa Ala Asn Gly Cys Arg Ser Lys His Met 6065 70 ctg cag cat cat att att gga gac naa nat tgt cta tac cta aac gtn352 Leu Gln His His Ile Ile Gly Asp Xaa Xaa Cys Leu Tyr Leu Asn Val 7580 85 tat gtt cca ttg act tcc aaa ttg gag aaa cta cca gta atg ttc tgg g401 Tyr Val Pro Leu Thr Ser Lys Leu Glu Lys Leu Pro Val Met Phe Trp 9095 100 2 103 PRT Ctenocephalides felis misc_feature (43)..(43) The ′Xaa′at location 43 stands for Lys, Arg, Thr, or Ile. 2 Met Asn Ser Leu IleVal Lys Ile Ser Gln Gly Ala Ile Glu Gly Lys 1 5 10 15 Glu Met Ile AsnAsp Asn Gly Lys Ser Phe Arg Gly Phe Leu Gly Ile 20 25 30 Pro Tyr Ala LysPro Pro Ile Gly Asn Leu Xaa Phe Lys Pro Pro Gln 35 40 45 Lys Pro Asp AspTrp Asn Asp Val Arg Pro Ala Thr Glu Xaa Ala Asn 50 55 60 Gly Cys Arg SerLys His Met Leu Gln His His Ile Ile Gly Asp Xaa 65 70 75 80 Xaa Cys LeuTyr Leu Asn Val Tyr Val Pro Leu Thr Ser Lys Leu Glu 85 90 95 Lys Leu ProVal Met Phe Trp 100 3 401 DNA Ctenocephalides felis misc_feature(50)..(50) n = unknown 3 cccagaacat tactggtagt ttctccaatt tggaagtcaatggaacatan acgtttaggt 60 atagacaatn ttngtctcca ataatatgat gctgcagcatatgtttcgat ctacaaccat 120 ttgcttnttc agtagctggt cgaacatcat tccaatcatcaggcttttga ggaggcttaa 180 atntaagatt tcctataggc ggtttagcat aaggtatacccaaaaatcct ctaaacgact 240 ttccattatc attaatcatt tccttcccct caatagctccttgagaaatt tttacaatta 300 aagaattcat tttgaaatat ttcacaaatc caacactctcactaataaac ttgatcttga 360 tccacaagat ttattagatt tatgtttatt aatgatgtaa a401 4 364 DNA Ctenocephalides felis CDS (2)..(364) 4 g tct cgt gtt attttt tta agt tgt att ttt ttg ttt agt ttt aat ttt 49 Ser Arg Val Ile PheLeu Ser Cys Ile Phe Leu Phe Ser Phe Asn Phe 1 5 10 15 ata aac tgt gattcc ccg act gta act ttg ccc caa ggc gaa ttg gtt 97 Ile Asn Cys Asp SerPro Thr Val Thr Leu Pro Gln Gly Glu Leu Val 20 25 30 gga aaa gct ttg acgaac gaa aat gga aaa gag tat ttt agc tac aca 145 Gly Lys Ala Leu Thr AsnGlu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr 35 40 45 ggt gta cct tat gct aaacct cct gtt gga gaa ctt aga ttt aag cct 193 Gly Val Pro Tyr Ala Lys ProPro Val Gly Glu Leu Arg Phe Lys Pro 50 55 60 cca cag aaa gct gag cca tggcaa ggt gtt ttc aac gcc aca tta tac 241 Pro Gln Lys Ala Glu Pro Trp GlnGly Val Phe Asn Ala Thr Leu Tyr 65 70 75 80 gga aat gtg tgt aaa tct ttaaat ttc ttc ttg aag aaa att gaa gga 289 Gly Asn Val Cys Lys Ser Leu AsnPhe Phe Leu Lys Lys Ile Glu Gly 85 90 95 gac gaa gac tgc ttg gta gta aacgtg tac gca cca aaa aca act tct 337 Asp Glu Asp Cys Leu Val Val Asn ValTyr Ala Pro Lys Thr Thr Ser 100 105 110 gat aaa aaa ctt cca gta ttt ttctgg 364 Asp Lys Lys Leu Pro Val Phe Phe Trp 115 120 5 121 PRTCtenocephalides felis 5 Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu PheSer Phe Asn Phe 1 5 10 15 Ile Asn Cys Asp Ser Pro Thr Val Thr Leu ProGln Gly Glu Leu Val 20 25 30 Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys GluTyr Phe Ser Tyr Thr 35 40 45 Gly Val Pro Tyr Ala Lys Pro Pro Val Gly GluLeu Arg Phe Lys Pro 50 55 60 Pro Gln Lys Ala Glu Pro Trp Gln Gly Val PheAsn Ala Thr Leu Tyr 65 70 75 80 Gly Asn Val Cys Lys Ser Leu Asn Phe PheLeu Lys Lys Ile Glu Gly 85 90 95 Asp Glu Asp Cys Leu Val Val Asn Val TyrAla Pro Lys Thr Thr Ser 100 105 110 Asp Lys Lys Leu Pro Val Phe Phe Trp115 120 6 364 DNA Ctenocephalides felis 6 ccagaaaaat actggaagttttttatcaga agttgttttt ggtgcgtaca cgtttactac 60 caagcagtct tcgtctccttcaattttctt caagaagaaa tttaaagatt tacacacatt 120 tccgtataat gtggcgttgaaaacaccttg ccatggctca gctttctgtg gaggcttaaa 180 tctaagttct ccaacaggaggtttagcata aggtacacct gtgtagctaa aatactcttt 240 tccattttcg ttcgtcaaagcttttccaac caattcgcct tggggcaaag ttacagtcgg 300 ggaatcacag tttataaaattaaaactaaa caaaaaaata caacttaaaa aaataacacg 360 agac 364 7 421 DNACtenocephalides felis CDS (113)..(421) 7 tttacattac atcaaatcatatttttatta gtatattttt tagaagaacc tagccaaaaa 60 atatggactt tagactgtgattaatttatt ttacctgaga ttttccttta ca atg ggt 118 Met Gly 1 gat ctt caagtg act ttg tta caa ggt tct ttg aga gga aaa gag caa 166 Asp Leu Gln ValThr Leu Leu Gln Gly Ser Leu Arg Gly Lys Glu Gln 5 10 15 att aat gaa aaggga aat gtg ttt tat agt tat tct gga att cca tat 214 Ile Asn Glu Lys GlyAsn Val Phe Tyr Ser Tyr Ser Gly Ile Pro Tyr 20 25 30 gcc aaa cct cca gttggt gat cta aga ttc aag cca cct caa cct gca 262 Ala Lys Pro Pro Val GlyAsp Leu Arg Phe Lys Pro Pro Gln Pro Ala 35 40 45 50 gaa cct tgg tca ggtgtc ctt gat gct act aaa gaa ggg aat agt tgt 310 Glu Pro Trp Ser Gly ValLeu Asp Ala Thr Lys Glu Gly Asn Ser Cys 55 60 65 aga tct gta cat ttt attaaa aag att aaa gta ggg gct gaa gat tgt 358 Arg Ser Val His Phe Ile LysLys Ile Lys Val Gly Ala Glu Asp Cys 70 75 80 cta tac ctc aat gtc tat gtacca aaa aca tca gag aaa tcc ctt ctt 406 Leu Tyr Leu Asn Val Tyr Val ProLys Thr Ser Glu Lys Ser Leu Leu 85 90 95 cca gta atg gta tgg 421 Pro ValMet Val Trp 100 8 103 PRT Ctenocephalides felis 8 Met Gly Asp Leu GlnVal Thr Leu Leu Gln Gly Ser Leu Arg Gly Lys 1 5 10 15 Glu Gln Ile AsnGlu Lys Gly Asn Val Phe Tyr Ser Tyr Ser Gly Ile 20 25 30 Pro Tyr Ala LysPro Pro Val Gly Asp Leu Arg Phe Lys Pro Pro Gln 35 40 45 Pro Ala Glu ProTrp Ser Gly Val Leu Asp Ala Thr Lys Glu Gly Asn 50 55 60 Ser Cys Arg SerVal His Phe Ile Lys Lys Ile Lys Val Gly Ala Glu 65 70 75 80 Asp Cys LeuTyr Leu Asn Val Tyr Val Pro Lys Thr Ser Glu Lys Ser 85 90 95 Leu Leu ProVal Met Val Trp 100 9 421 DNA Ctenocephalides felis 9 ccataccattactggaagaa gggatttctc tgatgttttt ggtacataga cattgaggta 60 tagacaatcttcagccccta ctttaatctt tttaataaaa tgtacagatc tacaactatt 120 cccttctttagtagcatcaa ggacacctga ccaaggttct gcaggttgag gtggcttgaa 180 tcttagatcaccaactggag gtttggcata tggaattcca gaataactat aaaacacatt 240 tcccttttcattaatttgct cttttcctct caaagaacct tgtaacaaag tcacttgaag 300 atcacccattgtaaaggaaa atctcaggta aaataaatta atcacagtct aaagtccata 360 ttttttggctaggttcttct aaaaaatata ctaataaaaa tatgatttga tgtaatgtaa 420 a 421 10 524DNA Ctenocephalides felis CDS (113)..(523) 10 gaacgttgat acgatagacatgtcgtcttc aaaacgtcta ttttatcata aacaaaacga 60 gataaataat aacaattaagcaaccaaaat gcattaaaaa acacaataaa aa atg tta 118 Met Leu 1 cct cac agtagt gca tta gtt tta ttt tta ttt ttt tta ttt ttc tta 166 Pro His Ser SerAla Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu 5 10 15 ttt aca cct atcttg tgc ata cta tgg gat aac cta gat cag cat ttg 214 Phe Thr Pro Ile LeuCys Ile Leu Trp Asp Asn Leu Asp Gln His Leu 20 25 30 tgc aga gtt caa tttaac agg atc acg gaa gga aaa ccg ttc cga tat 262 Cys Arg Val Gln Phe AsnArg Ile Thr Glu Gly Lys Pro Phe Arg Tyr 35 40 45 50 aaa gat cat agg aatgat gta tat tgt tct tat ttg gga att cct tat 310 Lys Asp His Arg Asn AspVal Tyr Cys Ser Tyr Leu Gly Ile Pro Tyr 55 60 65 gcc gaa ccg cct att ggacca tta cga ttt cag tct cca aaa cca ata 358 Ala Glu Pro Pro Ile Gly ProLeu Arg Phe Gln Ser Pro Lys Pro Ile 70 75 80 tca aat cca aaa aca gga ttcgta cag gct cga act ttg gga gac aaa 406 Ser Asn Pro Lys Thr Gly Phe ValGln Ala Arg Thr Leu Gly Asp Lys 85 90 95 tgt ttc cag gaa agt cta ata tattct tat gca gga agc gaa gat tgc 454 Cys Phe Gln Glu Ser Leu Ile Tyr SerTyr Ala Gly Ser Glu Asp Cys 100 105 110 tta tat ctg aat ata ttc acg ccagag act gtt aat tct gcg aac aat 502 Leu Tyr Leu Asn Ile Phe Thr Pro GluThr Val Asn Ser Ala Asn Asn 115 120 125 130 aca aaa tat cct gta atg ttct 524 Thr Lys Tyr Pro Val Met Phe 135 11 137 PRT Ctenocephalides felis11 Met Leu Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe 1 510 15 Phe Leu Phe Thr Pro Ile Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln 2025 30 His Leu Cys Arg Val Gln Phe Asn Arg Ile Thr Glu Gly Lys Pro Phe 3540 45 Arg Tyr Lys Asp His Arg Asn Asp Val Tyr Cys Ser Tyr Leu Gly Ile 5055 60 Pro Tyr Ala Glu Pro Pro Ile Gly Pro Leu Arg Phe Gln Ser Pro Lys 6570 75 80 Pro Ile Ser Asn Pro Lys Thr Gly Phe Val Gln Ala Arg Thr Leu Gly85 90 95 Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr Ser Tyr Ala Gly Ser Glu100 105 110 Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro Glu Thr Val Asn SerAla 115 120 125 Asn Asn Thr Lys Tyr Pro Val Met Phe 130 135 12 524 DNACtenocephalides felis 12 agaacattac aggatatttt gtattgttcg cagaattaacagtctctggc gtgaatatat 60 tcagatataa gcaatcttcg cttcctgcat aagaatatattagactttcc tggaaacatt 120 tgtctcccaa agttcgagcc tgtacgaatc ctgtttttggatttgatatt ggttttggag 180 actgaaatcg taatggtcca ataggcggtt cggcataaggaattcccaaa taagaacaat 240 atacatcatt cctatgatct ttatatcgga acggttttccttccgtgatc ctgttaaatt 300 gaactctgca caaatgctga tctaggttat cccatagtatgcacaagata ggtgtaaata 360 agaaaaataa aaaaaataaa aataaaacta atgcactactgtgaggtaac attttttatt 420 gtgtttttta atgcattttg gttgcttaat tgttattatttatctcgttt tgtttatgat 480 aaaatagacg ttttgaagac gacatgtcta tcgtatcaacgttc 524 13 1982 DNA Ctenocephalides felis CDS (3)..(1517) 13 at ttt agctac aca ggt gta cct tat gct aaa cct cct gtt gga gaa 47 Phe Ser Tyr ThrGly Val Pro Tyr Ala Lys Pro Pro Val Gly Glu 1 5 10 15 ctt aga ttt aagcct cca cag aaa gct gag cca tgg caa ggt gtt ttc 95 Leu Arg Phe Lys ProPro Gln Lys Ala Glu Pro Trp Gln Gly Val Phe 20 25 30 aac gcc aca tta tacgga aat gtg tgt aaa tct tta aat ttc ttc ttg 143 Asn Ala Thr Leu Tyr GlyAsn Val Cys Lys Ser Leu Asn Phe Phe Leu 35 40 45 aag aaa att gaa gga gacgaa gac tgc ttg gta gta aac gtg tac gca 191 Lys Lys Ile Glu Gly Asp GluAsp Cys Leu Val Val Asn Val Tyr Ala 50 55 60 cca aaa aca act tct gat aaaaaa ctt cca gta ttt ttc tgg gtt cat 239 Pro Lys Thr Thr Ser Asp Lys LysLeu Pro Val Phe Phe Trp Val His 65 70 75 ggt ggt ggt ttt gtg act gga tccgga aat tta gaa ttc caa agc cca 287 Gly Gly Gly Phe Val Thr Gly Ser GlyAsn Leu Glu Phe Gln Ser Pro 80 85 90 95 gat tat tta gta rat ttt gat gttatt ttc gta act ttc aat tac cga 335 Asp Tyr Leu Val Xaa Phe Asp Val IlePhe Val Thr Phe Asn Tyr Arg 100 105 110 ttg gga cct ctc gga ttt ctg aatttg gag ttg gag ggt gct cca gga 383 Leu Gly Pro Leu Gly Phe Leu Asn LeuGlu Leu Glu Gly Ala Pro Gly 115 120 125 aat gta gga tta ttg gat cag gtggca gct ctg aaa tgg acc aaa gaa 431 Asn Val Gly Leu Leu Asp Gln Val AlaAla Leu Lys Trp Thr Lys Glu 130 135 140 aac att gag aaa ttt ggt gga gatcca gaa aat att aca att ggt ggt 479 Asn Ile Glu Lys Phe Gly Gly Asp ProGlu Asn Ile Thr Ile Gly Gly 145 150 155 gtt tct gct ggt gga gca agt gttcat tat ctt ttg tta tct cat aca 527 Val Ser Ala Gly Gly Ala Ser Val HisTyr Leu Leu Leu Ser His Thr 160 165 170 175 acc act gga ctt tac aaa agggca att gct caa agt gga agt gct ttt 575 Thr Thr Gly Leu Tyr Lys Arg AlaIle Ala Gln Ser Gly Ser Ala Phe 180 185 190 aat cca tgg gcc ttc caa agacat cca gta aag cgt agt ctt caa ctt 623 Asn Pro Trp Ala Phe Gln Arg HisPro Val Lys Arg Ser Leu Gln Leu 195 200 205 gct gag ata ttg ggt cat cccaca aac aat act caa gat gct tta gaa 671 Ala Glu Ile Leu Gly His Pro ThrAsn Asn Thr Gln Asp Ala Leu Glu 210 215 220 ttc tta caa aaa gcc ccc gtagac agt ctc ctg aag aaa atg cca gct 719 Phe Leu Gln Lys Ala Pro Val AspSer Leu Leu Lys Lys Met Pro Ala 225 230 235 gaa aca gaa ggt gaa ata atagaa gag ttt gtc ttc gta cca tca att 767 Glu Thr Glu Gly Glu Ile Ile GluGlu Phe Val Phe Val Pro Ser Ile 240 245 250 255 gaa aaa gtt ttc cca tcccac caa cct ttc ttg gaa gaa tca cca ttg 815 Glu Lys Val Phe Pro Ser HisGln Pro Phe Leu Glu Glu Ser Pro Leu 260 265 270 gcc aga atg aaa tcc ggatcc ttt aac aaa gta cct tta tta gtt gga 863 Ala Arg Met Lys Ser Gly SerPhe Asn Lys Val Pro Leu Leu Val Gly 275 280 285 ttt aac agt gca gaa ggactt ttg ttc aaa ttc ttc atg aaa gaa aaa 911 Phe Asn Ser Ala Glu Gly LeuLeu Phe Lys Phe Phe Met Lys Glu Lys 290 295 300 cca gag atg ctg aac caagct gaa gca gat ttt gaa aga ctc gta cca 959 Pro Glu Met Leu Asn Gln AlaGlu Ala Asp Phe Glu Arg Leu Val Pro 305 310 315 gcc gaa ttt gaa tta gtccat gga tca gag gaa tcg aaa aaa ctt gca 1007 Ala Glu Phe Glu Leu Val HisGly Ser Glu Glu Ser Lys Lys Leu Ala 320 325 330 335 gaa aaa atc agg aagttt tac ttt gac gat aaa ccc gtt cca gaa aat 1055 Glu Lys Ile Arg Lys PheTyr Phe Asp Asp Lys Pro Val Pro Glu Asn 340 345 350 gaa cag aaa ttt attgac ttg ata gga gat att tgg ttt act aga ggt 1103 Glu Gln Lys Phe Ile AspLeu Ile Gly Asp Ile Trp Phe Thr Arg Gly 355 360 365 gtt gac aag cat gtcaag ttg tct gtg gag aaa caa gac gaa cca gtt 1151 Val Asp Lys His Val LysLeu Ser Val Glu Lys Gln Asp Glu Pro Val 370 375 380 tat tat tat gaa tattcc ttc tcg gaa agt cat cct gca aaa gga aca 1199 Tyr Tyr Tyr Glu Tyr SerPhe Ser Glu Ser His Pro Ala Lys Gly Thr 385 390 395 ttt ggt gat cat aatctg act ggt gca tgc cat gga gaa gaa ctt gtg 1247 Phe Gly Asp His Asn LeuThr Gly Ala Cys His Gly Glu Glu Leu Val 400 405 410 415 aat tta ttc aaagtc gag atg atg aag ctg gaa aaa gat aaa cct aat 1295 Asn Leu Phe Lys ValGlu Met Met Lys Leu Glu Lys Asp Lys Pro Asn 420 425 430 gtt cta tta acaaaa gat aga gta ctt gcc atg tgg act aac ttc atc 1343 Val Leu Leu Thr LysAsp Arg Val Leu Ala Met Trp Thr Asn Phe Ile 435 440 445 aaa aat gga aatcct act cct gaa gta aca gaa tta ttg cca gtt aaa 1391 Lys Asn Gly Asn ProThr Pro Glu Val Thr Glu Leu Leu Pro Val Lys 450 455 460 tgg gaa cct gccaca aaa gac aag ttg aat tat ttg aac att gat gcc 1439 Trp Glu Pro Ala ThrLys Asp Lys Leu Asn Tyr Leu Asn Ile Asp Ala 465 470 475 acc tta act ttggga aca aat cct gag gca aac cga gtc aaa ttt tgg 1487 Thr Leu Thr Leu GlyThr Asn Pro Glu Ala Asn Arg Val Lys Phe Trp 480 485 490 495 gaa gac gccaca aaa tct ttg cac ggt caa taataattta tgaaaattgt 1537 Glu Asp Ala ThrLys Ser Leu His Gly Gln 500 505 tttaaatact ttaggtaata tattaggtaaataaaaatta aaaaataaca atttttatgt 1597 tttatgtatt ggcttatgtg tatcagttctaattttattt atttattctt gttttgcttg 1657 ttttgaaata tcatggtttt aattttcaaaacacaacgtc gtttgttttt agcaaaattt 1717 ccaatagata tgttatatta agtactctgaagtattttta tatatacact aaaatcagta 1777 aaaatacatt aactaaaaat ataagatattttcaataatt ttttttaaag aaaataccaa 1837 aaataaagta aaattccaaa cggaatttttgtttaactta aaaataaaat taactcttca 1897 ataattttga taattagtat ttctgatatcattagtgaaa attatatttt gataatacgt 1957 atttatattt aaaataaaat tatgt 198214 505 PRT Ctenocephalides felis misc_feature (100)..(100) The ′Xaa′ atlocation 100 stands for Asp, or Asn. 14 Phe Ser Tyr Thr Gly Val Pro TyrAla Lys Pro Pro Val Gly Glu Leu 1 5 10 15 Arg Phe Lys Pro Pro Gln LysAla Glu Pro Trp Gln Gly Val Phe Asn 20 25 30 Ala Thr Leu Tyr Gly Asn ValCys Lys Ser Leu Asn Phe Phe Leu Lys 35 40 45 Lys Ile Glu Gly Asp Glu AspCys Leu Val Val Asn Val Tyr Ala Pro 50 55 60 Lys Thr Thr Ser Asp Lys LysLeu Pro Val Phe Phe Trp Val His Gly 65 70 75 80 Gly Gly Phe Val Thr GlySer Gly Asn Leu Glu Phe Gln Ser Pro Asp 85 90 95 Tyr Leu Val Xaa Phe AspVal Ile Phe Val Thr Phe Asn Tyr Arg Leu 100 105 110 Gly Pro Leu Gly PheLeu Asn Leu Glu Leu Glu Gly Ala Pro Gly Asn 115 120 125 Val Gly Leu LeuAsp Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn 130 135 140 Ile Glu LysPhe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly Val 145 150 155 160 SerAla Gly Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr Thr 165 170 175Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe Asn 180 185190 Pro Trp Ala Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala 195200 205 Glu Ile Leu Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe210 215 220 Leu Gln Lys Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro AlaGlu 225 230 235 240 Thr Glu Gly Glu Ile Ile Glu Glu Phe Val Phe Val ProSer Ile Glu 245 250 255 Lys Val Phe Pro Ser His Gln Pro Phe Leu Glu GluSer Pro Leu Ala 260 265 270 Arg Met Lys Ser Gly Ser Phe Asn Lys Val ProLeu Leu Val Gly Phe 275 280 285 Asn Ser Ala Glu Gly Leu Leu Phe Lys PhePhe Met Lys Glu Lys Pro 290 295 300 Glu Met Leu Asn Gln Ala Glu Ala AspPhe Glu Arg Leu Val Pro Ala 305 310 315 320 Glu Phe Glu Leu Val His GlySer Glu Glu Ser Lys Lys Leu Ala Glu 325 330 335 Lys Ile Arg Lys Phe TyrPhe Asp Asp Lys Pro Val Pro Glu Asn Glu 340 345 350 Gln Lys Phe Ile AspLeu Ile Gly Asp Ile Trp Phe Thr Arg Gly Val 355 360 365 Asp Lys His ValLys Leu Ser Val Glu Lys Gln Asp Glu Pro Val Tyr 370 375 380 Tyr Tyr GluTyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr Phe 385 390 395 400 GlyAsp His Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val Asn 405 410 415Leu Phe Lys Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn Val 420 425430 Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe Ile Lys 435440 445 Asn Gly Asn Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val Lys Trp450 455 460 Glu Pro Ala Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp AlaThr 465 470 475 480 Leu Thr Leu Gly Thr Asn Pro Glu Ala Asn Arg Val LysPhe Trp Glu 485 490 495 Asp Ala Thr Lys Ser Leu His Gly Gln 500 505 151982 DNA Ctenocephalides felis 15 acataatttt attttaaata taaatacgtattatcaaaat ataattttca ctaatgatat 60 cagaaatact aattatcaaa attattgaagagttaatttt atttttaagt taaacaaaaa 120 ttccgtttgg aattttactt tatttttggtattttcttta aaaaaaatta ttgaaaatat 180 cttatatttt tagttaatgt atttttactgattttagtgt atatataaaa atacttcaga 240 gtacttaata taacatatct attggaaattttgctaaaaa caaacgacgt tgtgttttga 300 aaattaaaac catgatattt caaaacaagcaaaacaagaa taaataaata aaattagaac 360 tgatacacat aagccaatac ataaaacataaaaattgtta ttttttaatt tttatttacc 420 taatatatta cctaaagtat ttaaaacaattttcataaat tattattgac cgtgcaaaga 480 ttttgtggcg tcttcccaaa atttgactcggtttgcctca ggatttgttc ccaaagttaa 540 ggtggcatca atgttcaaat aattcaacttgtcttttgtg gcaggttccc atttaactgg 600 caataattct gttacttcag gagtaggatttccatttttg atgaagttag tccacatggc 660 aagtactcta tcttttgtta atagaacattaggtttatct ttttccagct tcatcatctc 720 gactttgaat aaattcacaa gttcttctccatggcatgca ccagtcagat tatgatcacc 780 aaatgttcct tttgcaggat gactttccgagaaggaatat tcataataat aaactggttc 840 gtcttgtttc tccacagaca acttgacatgcttgtcaaca cctctagtaa accaaatatc 900 tcctatcaag tcaataaatt tctgttcattttctggaacg ggtttatcgt caaagtaaaa 960 cttcctgatt ttttctgcaa gttttttcgattcctctgat ccatggacta attcaaattc 1020 ggctggtacg agtctttcaa aatctgcttcagcttggttc agcatctctg gtttttcttt 1080 catgaagaat ttgaacaaaa gtccttctgcactgttaaat ccaactaata aaggtacttt 1140 gttaaaggat ccggatttca ttctggccaatggtgattct tccaagaaag gttggtggga 1200 tgggaaaact ttttcaattg atggtacgaagacaaactct tctattattt caccttctgt 1260 ttcagctggc attttcttca ggagactgtctacgggggct ttttgtaaga attctaaagc 1320 atcttgagta ttgtttgtgg gatgacccaatatctcagca agttgaagac tacgctttac 1380 tggatgtctt tggaaggccc atggattaaaagcacttcca ctttgagcaa ttgccctttt 1440 gtaaagtcca gtggttgtat gagataacaaaagataatga acacttgctc caccagcaga 1500 aacaccacca attgtaatat tttctggatctccaccaaat ttctcaatgt tttctttggt 1560 ccatttcaga gctgccacct gatccaataatcctacattt cctggagcac cctccaactc 1620 caaattcaga aatccgagag gtcccaatcggtaattgaaa gttacgaaaa taacatcaaa 1680 atytactaaa taatctgggc tttggaattctaaatttccg gatccagtca caaaaccacc 1740 accatgaacc cagaaaaata ctggaagttttttatcagaa gttgtttttg gtgcgtacac 1800 gtttactacc aagcagtctt cgtctccttcaattttcttc aagaagaaat ttaaagattt 1860 acacacattt ccgtataatg tggcgttgaaaacaccttgc catggctcag ctttctgtgg 1920 aggcttaaat ctaagttctc caacaggaggtttagcataa ggtacacctg tgtagctaaa 1980 at 1982 16 1515 DNACtenocephalides felis exon (1)..(1515) 16 ttt agc tac aca ggt gta ccttat gct aaa cct cct gtt gga gaa ctt 48 Phe Ser Tyr Thr Gly Val Pro TyrAla Lys Pro Pro Val Gly Glu Leu 1 5 10 15 aga ttt aag cct cca cag aaagct gag cca tgg caa ggt gtt ttc aac 96 Arg Phe Lys Pro Pro Gln Lys AlaGlu Pro Trp Gln Gly Val Phe Asn 20 25 30 gcc aca tta tac gga aat gtg tgtaaa tct tta aat ttc ttc ttg aag 144 Ala Thr Leu Tyr Gly Asn Val Cys LysSer Leu Asn Phe Phe Leu Lys 35 40 45 aaa att gaa gga gac gaa gac tgc ttggta gta aac gtg tac gca cca 192 Lys Ile Glu Gly Asp Glu Asp Cys Leu ValVal Asn Val Tyr Ala Pro 50 55 60 aaa aca act tct gat aaa aaa ctt cca gtattt ttc tgg gtt cat ggt 240 Lys Thr Thr Ser Asp Lys Lys Leu Pro Val PhePhe Trp Val His Gly 65 70 75 80 ggt ggt ttt gtg act gga tcc gga aat ttagaa ttc caa agc cca gat 288 Gly Gly Phe Val Thr Gly Ser Gly Asn Leu GluPhe Gln Ser Pro Asp 85 90 95 tat tta gta rat ttt gat gtt att ttc gta actttc aat tac cga ttg 336 Tyr Leu Val Xaa Phe Asp Val Ile Phe Val Thr PheAsn Tyr Arg Leu 100 105 110 gga cct ctc gga ttt ctg aat ttg gag ttg gagggt gct cca gga aat 384 Gly Pro Leu Gly Phe Leu Asn Leu Glu Leu Glu GlyAla Pro Gly Asn 115 120 125 gta gga tta ttg gat cag gtg gca gct ctg aaatgg acc aaa gaa aac 432 Val Gly Leu Leu Asp Gln Val Ala Ala Leu Lys TrpThr Lys Glu Asn 130 135 140 att gag aaa ttt ggt gga gat cca gaa aat attaca att ggt ggt gtt 480 Ile Glu Lys Phe Gly Gly Asp Pro Glu Asn Ile ThrIle Gly Gly Val 145 150 155 160 tct gct ggt gga gca agt gtt cat tat cttttg tta tct cat aca acc 528 Ser Ala Gly Gly Ala Ser Val His Tyr Leu LeuLeu Ser His Thr Thr 165 170 175 act gga ctt tac aaa agg gca att gct caaagt gga agt gct ttt aat 576 Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln SerGly Ser Ala Phe Asn 180 185 190 cca tgg gcc ttc caa aga cat cca gta aagcgt agt ctt caa ctt gct 624 Pro Trp Ala Phe Gln Arg His Pro Val Lys ArgSer Leu Gln Leu Ala 195 200 205 gag ata ttg ggt cat ccc aca aac aat actcaa gat gct tta gaa ttc 672 Glu Ile Leu Gly His Pro Thr Asn Asn Thr GlnAsp Ala Leu Glu Phe 210 215 220 tta caa aaa gcc ccc gta gac agt ctc ctgaag aaa atg cca gct gaa 720 Leu Gln Lys Ala Pro Val Asp Ser Leu Leu LysLys Met Pro Ala Glu 225 230 235 240 aca gaa ggt gaa ata ata gaa gag tttgtc ttc gta cca tca att gaa 768 Thr Glu Gly Glu Ile Ile Glu Glu Phe ValPhe Val Pro Ser Ile Glu 245 250 255 aaa gtt ttc cca tcc cac caa cct ttcttg gaa gaa tca cca ttg gcc 816 Lys Val Phe Pro Ser His Gln Pro Phe LeuGlu Glu Ser Pro Leu Ala 260 265 270 aga atg aaa tcc gga tcc ttt aac aaagta cct tta tta gtt gga ttt 864 Arg Met Lys Ser Gly Ser Phe Asn Lys ValPro Leu Leu Val Gly Phe 275 280 285 aac agt gca gaa gga ctt ttg ttc aaattc ttc atg aaa gaa aaa cca 912 Asn Ser Ala Glu Gly Leu Leu Phe Lys PhePhe Met Lys Glu Lys Pro 290 295 300 gag atg ctg aac caa gct gaa gca gatttt gaa aga ctc gta cca gcc 960 Glu Met Leu Asn Gln Ala Glu Ala Asp PheGlu Arg Leu Val Pro Ala 305 310 315 320 gaa ttt gaa tta gtc cat gga tcagag gaa tcg aaa aaa ctt gca gaa 1008 Glu Phe Glu Leu Val His Gly Ser GluGlu Ser Lys Lys Leu Ala Glu 325 330 335 aaa atc agg aag ttt tac ttt gacgat aaa ccc gtt cca gaa aat gaa 1056 Lys Ile Arg Lys Phe Tyr Phe Asp AspLys Pro Val Pro Glu Asn Glu 340 345 350 cag aaa ttt att gac ttg ata ggagat att tgg ttt act aga ggt gtt 1104 Gln Lys Phe Ile Asp Leu Ile Gly AspIle Trp Phe Thr Arg Gly Val 355 360 365 gac aag cat gtc aag ttg tct gtggag aaa caa gac gaa cca gtt tat 1152 Asp Lys His Val Lys Leu Ser Val GluLys Gln Asp Glu Pro Val Tyr 370 375 380 tat tat gaa tat tcc ttc tcg gaaagt cat cct gca aaa gga aca ttt 1200 Tyr Tyr Glu Tyr Ser Phe Ser Glu SerHis Pro Ala Lys Gly Thr Phe 385 390 395 400 ggt gat cat aat ctg act ggtgca tgc cat gga gaa gaa ctt gtg aat 1248 Gly Asp His Asn Leu Thr Gly AlaCys His Gly Glu Glu Leu Val Asn 405 410 415 tta ttc aaa gtc gag atg atgaag ctg gaa aaa gat aaa cct aat gtt 1296 Leu Phe Lys Val Glu Met Met LysLeu Glu Lys Asp Lys Pro Asn Val 420 425 430 cta tta aca aaa gat aga gtactt gcc atg tgg act aac ttc atc aaa 1344 Leu Leu Thr Lys Asp Arg Val LeuAla Met Trp Thr Asn Phe Ile Lys 435 440 445 aat gga aat cct act cct gaagta aca gaa tta ttg cca gtt aaa tgg 1392 Asn Gly Asn Pro Thr Pro Glu ValThr Glu Leu Leu Pro Val Lys Trp 450 455 460 gaa cct gcc aca aaa gac aagttg aat tat ttg aac att gat gcc acc 1440 Glu Pro Ala Thr Lys Asp Lys LeuAsn Tyr Leu Asn Ile Asp Ala Thr 465 470 475 480 tta act ttg gga aca aatcct gag gca aac cga gtc aaa ttt tgg gaa 1488 Leu Thr Leu Gly Thr Asn ProGlu Ala Asn Arg Val Lys Phe Trp Glu 485 490 495 gac gcc aca aaa tct ttgcac ggt caa 1515 Asp Ala Thr Lys Ser Leu His Gly Gln 500 505 17 1515 DNACtenocephalides felis 17 ttgaccgtgc aaagattttg tggcgtcttc ccaaaatttgactcggtttg cctcaggatt 60 tgttcccaaa gttaaggtgg catcaatgtt caaataattcaacttgtctt ttgtggcagg 120 ttcccattta actggcaata attctgttac ttcaggagtaggatttccat ttttgatgaa 180 gttagtccac atggcaagta ctctatcttt tgttaatagaacattaggtt tatctttttc 240 cagcttcatc atctcgactt tgaataaatt cacaagttcttctccatggc atgcaccagt 300 cagattatga tcaccaaatg ttccttttgc aggatgactttccgagaagg aatattcata 360 ataataaact ggttcgtctt gtttctccac agacaacttgacatgcttgt caacacctct 420 agtaaaccaa atatctccta tcaagtcaat aaatttctgttcattttctg gaacgggttt 480 atcgtcaaag taaaacttcc tgattttttc tgcaagttttttcgattcct ctgatccatg 540 gactaattca aattcggctg gtacgagtct ttcaaaatctgcttcagctt ggttcagcat 600 ctctggtttt tctttcatga agaatttgaa caaaagtccttctgcactgt taaatccaac 660 taataaaggt actttgttaa aggatccgga tttcattctggccaatggtg attcttccaa 720 gaaaggttgg tgggatggga aaactttttc aattgatggtacgaagacaa actcttctat 780 tatttcacct tctgtttcag ctggcatttt cttcaggagactgtctacgg gggctttttg 840 taagaattct aaagcatctt gagtattgtt tgtgggatgacccaatatct cagcaagttg 900 aagactacgc tttactggat gtctttggaa ggcccatggattaaaagcac ttccactttg 960 agcaattgcc cttttgtaaa gtccagtggt tgtatgagataacaaaagat aatgaacact 1020 tgctccacca gcagaaacac caccaattgt aatattttctggatctccac caaatttctc 1080 aatgttttct ttggtccatt tcagagctgc cacctgatccaataatccta catttcctgg 1140 agcaccctcc aactccaaat tcagaaatcc gagaggtcccaatcggtaat tgaaagttac 1200 gaaaataaca tcaaaatyta ctaaataatc tgggctttggaattctaaat ttccggatcc 1260 agtcacaaaa ccaccaccat gaacccagaa aaatactggaagttttttat cagaagttgt 1320 ttttggtgcg tacacgttta ctaccaagca gtcttcgtctccttcaattt tcttcaagaa 1380 gaaatttaaa gatttacaca catttccgta taatgtggcgttgaaaacac cttgccatgg 1440 ctcagctttc tgtggaggct taaatctaag ttctccaacaggaggtttag cataaggtac 1500 acctgtgtag ctaaa 1515 18 1792 DNACtenocephalides felis CDS (49)..(1701) 18 actgtgtgct aataattcagtacacacagt caatagtcta gatccaag atg tct cgt 57 Met Ser Arg 1 gtt att ttttta agt tgt att ttt ttg ttt agt ttt aat ttt ata aaa 105 Val Ile Phe LeuSer Cys Ile Phe Leu Phe Ser Phe Asn Phe Ile Lys 5 10 15 tgt gat ccc ccgact gta act ttg ccc cag ggc gaa ttg gtt gga aaa 153 Cys Asp Pro Pro ThrVal Thr Leu Pro Gln Gly Glu Leu Val Gly Lys 20 25 30 35 gct ttg acg aacgaa aat gga aaa gag tat ttt agc tac aca ggt gtg 201 Ala Leu Thr Asn GluAsn Gly Lys Glu Tyr Phe Ser Tyr Thr Gly Val 40 45 50 cct tat gct aaa cctcca gtt gga gaa ctt aga ttt aag cct cca cag 249 Pro Tyr Ala Lys Pro ProVal Gly Glu Leu Arg Phe Lys Pro Pro Gln 55 60 65 aaa gct gag cca tgg aatggt gtt ttc aac gcc aca tca cat gga aat 297 Lys Ala Glu Pro Trp Asn GlyVal Phe Asn Ala Thr Ser His Gly Asn 70 75 80 gtg tgc aaa gct ttg aat ttcttc ttg aaa aaa att gaa gga gac gaa 345 Val Cys Lys Ala Leu Asn Phe PheLeu Lys Lys Ile Glu Gly Asp Glu 85 90 95 gac tgc ttg ttg gtg aat gtg tacgca cca aaa aca act tct gac aaa 393 Asp Cys Leu Leu Val Asn Val Tyr AlaPro Lys Thr Thr Ser Asp Lys 100 105 110 115 aaa ctt cca gta ttt ttc tgggtt cat ggt ggc ggt ttt gtg act gga 441 Lys Leu Pro Val Phe Phe Trp ValHis Gly Gly Gly Phe Val Thr Gly 120 125 130 tcc gga aat tta gaa ttt caaagc cca gat tat tta gta aat tat gat 489 Ser Gly Asn Leu Glu Phe Gln SerPro Asp Tyr Leu Val Asn Tyr Asp 135 140 145 gtt att ttt gta act ttc aattac cga ttg gga cca ctc gga ttt ttg 537 Val Ile Phe Val Thr Phe Asn TyrArg Leu Gly Pro Leu Gly Phe Leu 150 155 160 aat ttg gag ttg gaa ggt gctcct gga aat gta gga tta ttg gat cag 585 Asn Leu Glu Leu Glu Gly Ala ProGly Asn Val Gly Leu Leu Asp Gln 165 170 175 gta gca gct ttg aaa tgg accaaa gaa aat att gag aaa ttt ggt gga 633 Val Ala Ala Leu Lys Trp Thr LysGlu Asn Ile Glu Lys Phe Gly Gly 180 185 190 195 gat cca gaa aat att acaatt ggt ggt gtt tct gct ggt gga gca agt 681 Asp Pro Glu Asn Ile Thr IleGly Gly Val Ser Ala Gly Gly Ala Ser 200 205 210 gtt cat tat ctt tta ttgtca cat aca acc act gga ctt tac aaa agg 729 Val His Tyr Leu Leu Leu SerHis Thr Thr Thr Gly Leu Tyr Lys Arg 215 220 225 gca att gct caa agt ggaagt gct tta aat cca tgg gcc ttc caa aga 777 Ala Ile Ala Gln Ser Gly SerAla Leu Asn Pro Trp Ala Phe Gln Arg 230 235 240 cat cca gta aag cgt agtctt caa ctt gct gag ata tta ggt cat ccc 825 His Pro Val Lys Arg Ser LeuGln Leu Ala Glu Ile Leu Gly His Pro 245 250 255 aca aac aac act caa gatgct tta gaa ttc tta caa aaa gcc cca gta 873 Thr Asn Asn Thr Gln Asp AlaLeu Glu Phe Leu Gln Lys Ala Pro Val 260 265 270 275 gac agt ctc ctg aaaaaa atg cca gct gaa aca gaa ggt gaa ata ata 921 Asp Ser Leu Leu Lys LysMet Pro Ala Glu Thr Glu Gly Glu Ile Ile 280 285 290 gaa gag ttc gtc ttcgta cca tca att gaa aaa gtt ttc cca tcc cac 969 Glu Glu Phe Val Phe ValPro Ser Ile Glu Lys Val Phe Pro Ser His 295 300 305 caa cct ttc ttg gaagaa tca cca ttg gcc aga atg aaa tct gga tcc 1017 Gln Pro Phe Leu Glu GluSer Pro Leu Ala Arg Met Lys Ser Gly Ser 310 315 320 ttt aac aaa gta ccttta tta gtt gga ttc aac agc gca gaa gga ctt 1065 Phe Asn Lys Val Pro LeuLeu Val Gly Phe Asn Ser Ala Glu Gly Leu 325 330 335 ttg tac aaa ttc tttatg aaa gaa aaa cca gag atg ctg aac caa gct 1113 Leu Tyr Lys Phe Phe MetLys Glu Lys Pro Glu Met Leu Asn Gln Ala 340 345 350 355 gaa gca gat ttcgaa aga ctc gta cca gcc gaa ttt gaa tta gcc cat 1161 Glu Ala Asp Phe GluArg Leu Val Pro Ala Glu Phe Glu Leu Ala His 360 365 370 gga tca gaa gaatcg aaa aaa ctt gca gaa aaa atc agg aag ttt tac 1209 Gly Ser Glu Glu SerLys Lys Leu Ala Glu Lys Ile Arg Lys Phe Tyr 375 380 385 ttt gac gat aaaccc gtt cct gaa aat gag cag aaa ttt att gac ttg 1257 Phe Asp Asp Lys ProVal Pro Glu Asn Glu Gln Lys Phe Ile Asp Leu 390 395 400 ata gga gat atttgg ttt act aga ggc att gac aag cat gtc aag ttg 1305 Ile Gly Asp Ile TrpPhe Thr Arg Gly Ile Asp Lys His Val Lys Leu 405 410 415 tct gta gaa aaacaa gac gag cca gta tat tat tat gaa tat tct ttc 1353 Ser Val Glu Lys GlnAsp Glu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe 420 425 430 435 tct gaa agtcat cct gca aaa gga aca ttt ggt gac cat aac ttg act 1401 Ser Glu Ser HisPro Ala Lys Gly Thr Phe Gly Asp His Asn Leu Thr 440 445 450 gga gca tgtcat ggt gaa gaa ctt gtg aat tta ttc aaa gtc gag atg 1449 Gly Ala Cys HisGly Glu Glu Leu Val Asn Leu Phe Lys Val Glu Met 455 460 465 atg aag ctggaa aaa gat aaa ccg aat gtt tta tta aca aaa gat agg 1497 Met Lys Leu GluLys Asp Lys Pro Asn Val Leu Leu Thr Lys Asp Arg 470 475 480 gta ctt gctatg tgg acg aac ttc atc aaa aat gga aat cct act cct 1545 Val Leu Ala MetTrp Thr Asn Phe Ile Lys Asn Gly Asn Pro Thr Pro 485 490 495 gaa gta actgaa tta ttg cca gtt aaa tgg gaa cct gcc aca aaa gac 1593 Glu Val Thr GluLeu Leu Pro Val Lys Trp Glu Pro Ala Thr Lys Asp 500 505 510 515 aag ttgaat tat ttg aac att gat gcc acc tta act ttg gga aca aat 1641 Lys Leu AsnTyr Leu Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn 520 525 530 cca gaagaa acc cga gtc aaa tty tgg gaa gat gcc aca aaa act ttg 1689 Pro Glu GluThr Arg Val Lys Phe Trp Glu Asp Ala Thr Lys Thr Leu 535 540 545 cac agtcaa taa aaatgtatga aaattgtttt aattatttta ggtaatacat 1741 His Ser Gln 550taggtaaata aaaattnaaa aataacnaaa aaaaaaaaaa aaaaaaaaaa a 1792 19 550 PRTCtenocephalides felis 19 Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe LeuPhe Ser Phe Asn 1 5 10 15 Phe Ile Lys Cys Asp Pro Pro Thr Val Thr LeuPro Gln Gly Glu Leu 20 25 30 Val Gly Lys Ala Leu Thr Asn Glu Asn Gly LysGlu Tyr Phe Ser Tyr 35 40 45 Thr Gly Val Pro Tyr Ala Lys Pro Pro Val GlyGlu Leu Arg Phe Lys 50 55 60 Pro Pro Gln Lys Ala Glu Pro Trp Asn Gly ValPhe Asn Ala Thr Ser 65 70 75 80 His Gly Asn Val Cys Lys Ala Leu Asn PhePhe Leu Lys Lys Ile Glu 85 90 95 Gly Asp Glu Asp Cys Leu Leu Val Asn ValTyr Ala Pro Lys Thr Thr 100 105 110 Ser Asp Lys Lys Leu Pro Val Phe PheTrp Val His Gly Gly Gly Phe 115 120 125 Val Thr Gly Ser Gly Asn Leu GluPhe Gln Ser Pro Asp Tyr Leu Val 130 135 140 Asn Tyr Asp Val Ile Phe ValThr Phe Asn Tyr Arg Leu Gly Pro Leu 145 150 155 160 Gly Phe Leu Asn LeuGlu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu 165 170 175 Leu Asp Gln ValAla Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys 180 185 190 Phe Gly GlyAsp Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly 195 200 205 Gly AlaSer Val His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu 210 215 220 TyrLys Arg Ala Ile Ala Gln Ser Gly Ser Ala Leu Asn Pro Trp Ala 225 230 235240 Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu 245250 255 Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu Gln Lys260 265 270 Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro Ala Glu Thr GluGly 275 280 285 Glu Ile Ile Glu Glu Phe Val Phe Val Pro Ser Ile Glu LysVal Phe 290 295 300 Pro Ser His Gln Pro Phe Leu Glu Glu Ser Pro Leu AlaArg Met Lys 305 310 315 320 Ser Gly Ser Phe Asn Lys Val Pro Leu Leu ValGly Phe Asn Ser Ala 325 330 335 Glu Gly Leu Leu Tyr Lys Phe Phe Met LysGlu Lys Pro Glu Met Leu 340 345 350 Asn Gln Ala Glu Ala Asp Phe Glu ArgLeu Val Pro Ala Glu Phe Glu 355 360 365 Leu Ala His Gly Ser Glu Glu SerLys Lys Leu Ala Glu Lys Ile Arg 370 375 380 Lys Phe Tyr Phe Asp Asp LysPro Val Pro Glu Asn Glu Gln Lys Phe 385 390 395 400 Ile Asp Leu Ile GlyAsp Ile Trp Phe Thr Arg Gly Ile Asp Lys His 405 410 415 Val Lys Leu SerVal Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu 420 425 430 Tyr Ser PheSer Glu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His 435 440 445 Asn LeuThr Gly Ala Cys His Gly Glu Glu Leu Val Asn Leu Phe Lys 450 455 460 ValGlu Met Met Lys Leu Glu Lys Asp Lys Pro Asn Val Leu Leu Thr 465 470 475480 Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly Asn 485490 495 Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro Ala500 505 510 Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp Ala Thr Leu ThrLeu 515 520 525 Gly Thr Asn Pro Glu Glu Thr Arg Val Lys Phe Trp Glu AspAla Thr 530 535 540 Lys Thr Leu His Ser Gln 545 550 20 1792 DNACtenocephalides felis misc_feature (25)..(25) n = unknown 20 tttttttttttttttttttt ttttngttat ttttnaattt ttatttacct aatgtattac 60 ctaaaataattaaaacaatt ttcatacatt tttattgact gtgcaaagtt tttgtggcat 120 cttcccaraatttgactcgg gtttcttctg gatttgttcc caaagttaag gtggcatcaa 180 tgttcaaataattcaacttg tcttttgtgg caggttccca tttaactggc aataattcag 240 ttacttcaggagtaggattt ccatttttga tgaagttcgt ccacatagca agtaccctat 300 cttttgttaataaaacattc ggtttatctt tttccagctt catcatctcg actttgaata 360 aattcacaagttcttcacca tgacatgctc cagtcaagtt atggtcacca aatgttcctt 420 ttgcaggatgactttcagag aaagaatatt cataataata tactggctcg tcttgttttt 480 ctacagacaacttgacatgc ttgtcaatgc ctctagtaaa ccaaatatct cctatcaagt 540 caataaatttctgctcattt tcaggaacgg gtttatcgtc aaagtaaaac ttcctgattt 600 tttctgcaagttttttcgat tcttctgatc catgggctaa ttcaaattcg gctggtacga 660 gtctttcgaaatctgcttca gcttggttca gcatctctgg tttttctttc ataaagaatt 720 tgtacaaaagtccttctgcg ctgttgaatc caactaataa aggtactttg ttaaaggatc 780 cagatttcattctggccaat ggtgattctt ccaagaaagg ttggtgggat gggaaaactt 840 tttcaattgatggtacgaag acgaactctt ctattatttc accttctgtt tcagctggca 900 tttttttcaggagactgtct actggggctt tttgtaagaa ttctaaagca tcttgagtgt 960 tgtttgtgggatgacctaat atctcagcaa gttgaagact acgctttact ggatgtcttt 1020 ggaaggcccatggatttaaa gcacttccac tttgagcaat tgcccttttg taaagtccag 1080 tggttgtatgtgacaataaa agataatgaa cacttgctcc accagcagaa acaccaccaa 1140 ttgtaatattttctggatct ccaccaaatt tctcaatatt ttctttggtc catttcaaag 1200 ctgctacctgatccaataat cctacatttc caggagcacc ttccaactcc aaattcaaaa 1260 atccgagtggtcccaatcgg taattgaaag ttacaaaaat aacatcataa tttactaaat 1320 aatctgggctttgaaattct aaatttccgg atccagtcac aaaaccgcca ccatgaaccc 1380 agaaaaatactggaagtttt ttgtcagaag ttgtttttgg tgcgtacaca ttcaccaaca 1440 agcagtcttcgtctccttca atttttttca agaagaaatt caaagctttg cacacatttc 1500 catgtgatgtggcgttgaaa acaccattcc atggctcagc tttctgtgga ggcttaaatc 1560 taagttctccaactggaggt ttagcataag gcacacctgt gtagctaaaa tactcttttc 1620 cattttcgttcgtcaaagct tttccaacca attcgccctg gggcaaagtt acagtcgggg 1680 gatcacattttataaaatta aaactaaaca aaaaaataca acttaaaaaa ataacacgag 1740 acatcttggatctagactat tgactgtgtg tactgaatta ttagcacaca gt 1792 21 1650 DNACtenocephalides felis exon (1)..(1650) 21 atg tct cgt gtt att ttt ttaagt tgt att ttt ttg ttt agt ttt aat 48 Met Ser Arg Val Ile Phe Leu SerCys Ile Phe Leu Phe Ser Phe Asn 1 5 10 15 ttt ata aaa tgt gat ccc ccgact gta act ttg ccc cag ggc gaa ttg 96 Phe Ile Lys Cys Asp Pro Pro ThrVal Thr Leu Pro Gln Gly Glu Leu 20 25 30 gtt gga aaa gct ttg acg aac gaaaat gga aaa gag tat ttt agc tac 144 Val Gly Lys Ala Leu Thr Asn Glu AsnGly Lys Glu Tyr Phe Ser Tyr 35 40 45 aca ggt gtg cct tat gct aaa cct ccagtt gga gaa ctt aga ttt aag 192 Thr Gly Val Pro Tyr Ala Lys Pro Pro ValGly Glu Leu Arg Phe Lys 50 55 60 cct cca cag aaa gct gag cca tgg aat ggtgtt ttc aac gcc aca tca 240 Pro Pro Gln Lys Ala Glu Pro Trp Asn Gly ValPhe Asn Ala Thr Ser 65 70 75 80 cat gga aat gtg tgc aaa gct ttg aat ttcttc ttg aaa aaa att gaa 288 His Gly Asn Val Cys Lys Ala Leu Asn Phe PheLeu Lys Lys Ile Glu 85 90 95 gga gac gaa gac tgc ttg ttg gtg aat gtg tacgca cca aaa aca act 336 Gly Asp Glu Asp Cys Leu Leu Val Asn Val Tyr AlaPro Lys Thr Thr 100 105 110 tct gac aaa aaa ctt cca gta ttt ttc tgg gttcat ggt ggc ggt ttt 384 Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val HisGly Gly Gly Phe 115 120 125 gtg act gga tcc gga aat tta gaa ttt caa agccca gat tat tta gta 432 Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser ProAsp Tyr Leu Val 130 135 140 aat tat gat gtt att ttt gta act ttc aat taccga ttg gga cca ctc 480 Asn Tyr Asp Val Ile Phe Val Thr Phe Asn Tyr ArgLeu Gly Pro Leu 145 150 155 160 gga ttt ttg aat ttg gag ttg gaa ggt gctcct gga aat gta gga tta 528 Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala ProGly Asn Val Gly Leu 165 170 175 ttg gat cag gta gca gct ttg aaa tgg accaaa gaa aat att gag aaa 576 Leu Asp Gln Val Ala Ala Leu Lys Trp Thr LysGlu Asn Ile Glu Lys 180 185 190 ttt ggt gga gat cca gaa aat att aca attggt ggt gtt tct gct ggt 624 Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile GlyGly Val Ser Ala Gly 195 200 205 gga gca agt gtt cat tat ctt tta ttg tcacat aca acc act gga ctt 672 Gly Ala Ser Val His Tyr Leu Leu Leu Ser HisThr Thr Thr Gly Leu 210 215 220 tac aaa agg gca att gct caa agt gga agtgct tta aat cca tgg gcc 720 Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser AlaLeu Asn Pro Trp Ala 225 230 235 240 ttc caa aga cat cca gta aag cgt agtctt caa ctt gct gag ata tta 768 Phe Gln Arg His Pro Val Lys Arg Ser LeuGln Leu Ala Glu Ile Leu 245 250 255 ggt cat ccc aca aac aac act caa gatgct tta gaa ttc tta caa aaa 816 Gly His Pro Thr Asn Asn Thr Gln Asp AlaLeu Glu Phe Leu Gln Lys 260 265 270 gcc cca gta gac agt ctc ctg aaa aaaatg cca gct gaa aca gaa ggt 864 Ala Pro Val Asp Ser Leu Leu Lys Lys MetPro Ala Glu Thr Glu Gly 275 280 285 gaa ata ata gaa gag ttc gtc ttc gtacca tca att gaa aaa gtt ttc 912 Glu Ile Ile Glu Glu Phe Val Phe Val ProSer Ile Glu Lys Val Phe 290 295 300 cca tcc cac caa cct ttc ttg gaa gaatca cca ttg gcc aga atg aaa 960 Pro Ser His Gln Pro Phe Leu Glu Glu SerPro Leu Ala Arg Met Lys 305 310 315 320 tct gga tcc ttt aac aaa gta ccttta tta gtt gga ttc aac agc gca 1008 Ser Gly Ser Phe Asn Lys Val Pro LeuLeu Val Gly Phe Asn Ser Ala 325 330 335 gaa gga ctt ttg tac aaa ttc tttatg aaa gaa aaa cca gag atg ctg 1056 Glu Gly Leu Leu Tyr Lys Phe Phe MetLys Glu Lys Pro Glu Met Leu 340 345 350 aac caa gct gaa gca gat ttc gaaaga ctc gta cca gcc gaa ttt gaa 1104 Asn Gln Ala Glu Ala Asp Phe Glu ArgLeu Val Pro Ala Glu Phe Glu 355 360 365 tta gcc cat gga tca gaa gaa tcgaaa aaa ctt gca gaa aaa atc agg 1152 Leu Ala His Gly Ser Glu Glu Ser LysLys Leu Ala Glu Lys Ile Arg 370 375 380 aag ttt tac ttt gac gat aaa cccgtt cct gaa aat gag cag aaa ttt 1200 Lys Phe Tyr Phe Asp Asp Lys Pro ValPro Glu Asn Glu Gln Lys Phe 385 390 395 400 att gac ttg ata gga gat atttgg ttt act aga ggc att gac aag cat 1248 Ile Asp Leu Ile Gly Asp Ile TrpPhe Thr Arg Gly Ile Asp Lys His 405 410 415 gtc aag ttg tct gta gaa aaacaa gac gag cca gta tat tat tat gaa 1296 Val Lys Leu Ser Val Glu Lys GlnAsp Glu Pro Val Tyr Tyr Tyr Glu 420 425 430 tat tct ttc tct gaa agt catcct gca aaa gga aca ttt ggt gac cat 1344 Tyr Ser Phe Ser Glu Ser His ProAla Lys Gly Thr Phe Gly Asp His 435 440 445 aac ttg act gga gca tgt catggt gaa gaa ctt gtg aat tta ttc aaa 1392 Asn Leu Thr Gly Ala Cys His GlyGlu Glu Leu Val Asn Leu Phe Lys 450 455 460 gtc gag atg atg aag ctg gaaaaa gat aaa ccg aat gtt tta tta aca 1440 Val Glu Met Met Lys Leu Glu LysAsp Lys Pro Asn Val Leu Leu Thr 465 470 475 480 aaa gat agg gta ctt gctatg tgg acg aac ttc atc aaa aat gga aat 1488 Lys Asp Arg Val Leu Ala MetTrp Thr Asn Phe Ile Lys Asn Gly Asn 485 490 495 cct act cct gaa gta actgaa tta ttg cca gtt aaa tgg gaa cct gcc 1536 Pro Thr Pro Glu Val Thr GluLeu Leu Pro Val Lys Trp Glu Pro Ala 500 505 510 aca aaa gac aag ttg aattat ttg aac att gat gcc acc tta act ttg 1584 Thr Lys Asp Lys Leu Asn TyrLeu Asn Ile Asp Ala Thr Leu Thr Leu 515 520 525 gga aca aat cca gaa gaaacc cga gtc aaa tty tgg gaa gat gcc aca 1632 Gly Thr Asn Pro Glu Glu ThrArg Val Lys Phe Trp Glu Asp Ala Thr 530 535 540 aaa act ttg cac agt caa1650 Lys Thr Leu His Ser Gln 545 550 22 1650 DNA Ctenocephalides felis22 ttgactgtgc aaagtttttg tggcatcttc ccaraatttg actcgggttt cttctggatt 60tgttcccaaa gttaaggtgg catcaatgtt caaataattc aacttgtctt ttgtggcagg 120ttcccattta actggcaata attcagttac ttcaggagta ggatttccat ttttgatgaa 180gttcgtccac atagcaagta ccctatcttt tgttaataaa acattcggtt tatctttttc 240cagcttcatc atctcgactt tgaataaatt cacaagttct tcaccatgac atgctccagt 300caagttatgg tcaccaaatg ttccttttgc aggatgactt tcagagaaag aatattcata 360ataatatact ggctcgtctt gtttttctac agacaacttg acatgcttgt caatgcctct 420agtaaaccaa atatctccta tcaagtcaat aaatttctgc tcattttcag gaacgggttt 480atcgtcaaag taaaacttcc tgattttttc tgcaagtttt ttcgattctt ctgatccatg 540ggctaattca aattcggctg gtacgagtct ttcgaaatct gcttcagctt ggttcagcat 600ctctggtttt tctttcataa agaatttgta caaaagtcct tctgcgctgt tgaatccaac 660taataaaggt actttgttaa aggatccaga tttcattctg gccaatggtg attcttccaa 720gaaaggttgg tgggatggga aaactttttc aattgatggt acgaagacga actcttctat 780tatttcacct tctgtttcag ctggcatttt tttcaggaga ctgtctactg gggctttttg 840taagaattct aaagcatctt gagtgttgtt tgtgggatga cctaatatct cagcaagttg 900aagactacgc tttactggat gtctttggaa ggcccatgga tttaaagcac ttccactttg 960agcaattgcc cttttgtaaa gtccagtggt tgtatgtgac aataaaagat aatgaacact 1020tgctccacca gcagaaacac caccaattgt aatattttct ggatctccac caaatttctc 1080aatattttct ttggtccatt tcaaagctgc tacctgatcc aataatccta catttccagg 1140agcaccttcc aactccaaat tcaaaaatcc gagtggtccc aatcggtaat tgaaagttac 1200aaaaataaca tcataattta ctaaataatc tgggctttga aattctaaat ttccggatcc 1260agtcacaaaa ccgccaccat gaacccagaa aaatactgga agttttttgt cagaagttgt 1320ttttggtgcg tacacattca ccaacaagca gtcttcgtct ccttcaattt ttttcaagaa 1380gaaattcaaa gctttgcaca catttccatg tgatgtggcg ttgaaaacac cattccatgg 1440ctcagctttc tgtggaggct taaatctaag ttctccaact ggaggtttag cataaggcac 1500acctgtgtag ctaaaatact cttttccatt ttcgttcgtc aaagcttttc caaccaattc 1560gccctggggc aaagttacag tcgggggatc acattttata aaattaaaac taaacaaaaa 1620aatacaactt aaaaaaataa cacgagacat 1650 23 1590 DNA Ctenocephalides felisexon (1)..(1590) 23 gat ccc ccg act gta act ttg ccc cag ggc gaa ttg gttgga aaa gct 48 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val GlyLys Ala 1 5 10 15 ttg acg aac gaa aat gga aaa gag tat ttt agc tac acaggt gtg cct 96 Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr GlyVal Pro 20 25 30 tat gct aaa cct cca gtt gga gaa ctt aga ttt aag cct ccacag aaa 144 Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys Pro Pro GlnLys 35 40 45 gct gag cca tgg aat ggt gtt ttc aac gcc aca tca cat gga aatgtg 192 Ala Glu Pro Trp Asn Gly Val Phe Asn Ala Thr Ser His Gly Asn Val50 55 60 tgc aaa gct ttg aat ttc ttc ttg aaa aaa att gaa gga gac gaa gac240 Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp Glu Asp 6570 75 80 tgc ttg ttg gtg aat gtg tac gca cca aaa aca act tct gac aaa aaa288 Cys Leu Leu Val Asn Val Tyr Ala Pro Lys Thr Thr Ser Asp Lys Lys 8590 95 ctt cca gta ttt ttc tgg gtt cat ggt ggc ggt ttt gtg act gga tcc336 Leu Pro Val Phe Phe Trp Val His Gly Gly Gly Phe Val Thr Gly Ser 100105 110 gga aat tta gaa ttt caa agc cca gat tat tta gta aat tat gat gtt384 Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu Val Asn Tyr Asp Val 115120 125 att ttt gta act ttc aat tac cga ttg gga cca ctc gga ttt ttg aat432 Ile Phe Val Thr Phe Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu Asn 130135 140 ttg gag ttg gaa ggt gct cct gga aat gta gga tta ttg gat cag gta480 Leu Glu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Val 145150 155 160 gca gct ttg aaa tgg acc aaa gaa aat att gag aaa ttt ggt ggagat 528 Ala Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys Phe Gly Gly Asp165 170 175 cca gaa aat att aca att ggt ggt gtt tct gct ggt gga gca agtgtt 576 Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly Gly Ala Ser Val180 185 190 cat tat ctt tta ttg tca cat aca acc act gga ctt tac aaa agggca 624 His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg Ala195 200 205 att gct caa agt gga agt gct tta aat cca tgg gcc ttc caa agacat 672 Ile Ala Gln Ser Gly Ser Ala Leu Asn Pro Trp Ala Phe Gln Arg His210 215 220 cca gta aag cgt agt ctt caa ctt gct gag ata tta ggt cat cccaca 720 Pro Val Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu Gly His Pro Thr225 230 235 240 aac aac act caa gat gct tta gaa ttc tta caa aaa gcc ccagta gac 768 Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu Gln Lys Ala Pro ValAsp 245 250 255 agt ctc ctg aaa aaa atg cca gct gaa aca gaa ggt gaa ataata gaa 816 Ser Leu Leu Lys Lys Met Pro Ala Glu Thr Glu Gly Glu Ile IleGlu 260 265 270 gag ttc gtc ttc gta cca tca att gaa aaa gtt ttc cca tcccac caa 864 Glu Phe Val Phe Val Pro Ser Ile Glu Lys Val Phe Pro Ser HisGln 275 280 285 cct ttc ttg gaa gaa tca cca ttg gcc aga atg aaa tct ggatcc ttt 912 Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys Ser Gly SerPhe 290 295 300 aac aaa gta cct tta tta gtt gga ttc aac agc gca gaa ggactt ttg 960 Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala Glu Gly LeuLeu 305 310 315 320 tac aaa ttc ttt atg aaa gaa aaa cca gag atg ctg aaccaa gct gaa 1008 Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu Met Leu Asn GlnAla Glu 325 330 335 gca gat ttc gaa aga ctc gta cca gcc gaa ttt gaa ttagcc cat gga 1056 Ala Asp Phe Glu Arg Leu Val Pro Ala Glu Phe Glu Leu AlaHis Gly 340 345 350 tca gaa gaa tcg aaa aaa ctt gca gaa aaa atc agg aagttt tac ttt 1104 Ser Glu Glu Ser Lys Lys Leu Ala Glu Lys Ile Arg Lys PheTyr Phe 355 360 365 gac gat aaa ccc gtt cct gaa aat gag cag aaa ttt attgac ttg ata 1152 Asp Asp Lys Pro Val Pro Glu Asn Glu Gln Lys Phe Ile AspLeu Ile 370 375 380 gga gat att tgg ttt act aga ggc att gac aag cat gtcaag ttg tct 1200 Gly Asp Ile Trp Phe Thr Arg Gly Ile Asp Lys His Val LysLeu Ser 385 390 395 400 gta gaa aaa caa gac gag cca gta tat tat tat gaatat tct ttc tct 1248 Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu TyrSer Phe Ser 405 410 415 gaa agt cat cct gca aaa gga aca ttt ggt gac cataac ttg act gga 1296 Glu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His AsnLeu Thr Gly 420 425 430 gca tgt cat ggt gaa gaa ctt gtg aat tta ttc aaagtc gag atg atg 1344 Ala Cys His Gly Glu Glu Leu Val Asn Leu Phe Lys ValGlu Met Met 435 440 445 aag ctg gaa aaa gat aaa ccg aat gtt tta tta acaaaa gat agg gta 1392 Lys Leu Glu Lys Asp Lys Pro Asn Val Leu Leu Thr LysAsp Arg Val 450 455 460 ctt gct atg tgg acg aac ttc atc aaa aat gga aatcct act cct gaa 1440 Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly Asn ProThr Pro Glu 465 470 475 480 gta act gaa tta ttg cca gtt aaa tgg gaa cctgcc aca aaa gac aag 1488 Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro AlaThr Lys Asp Lys 485 490 495 ttg aat tat ttg aac att gat gcc acc tta actttg gga aca aat cca 1536 Leu Asn Tyr Leu Asn Ile Asp Ala Thr Leu Thr LeuGly Thr Asn Pro 500 505 510 gaa gaa acc cga gtc aaa tty tgg gaa gat gccaca aaa act ttg cac 1584 Glu Glu Thr Arg Val Lys Phe Trp Glu Asp Ala ThrLys Thr Leu His 515 520 525 agt caa 1590 Ser Gln 530 24 2836 DNACtenocephalides felis CDS (99)..(1889) 24 tagacatgtc gtcttcaaaacgtctatttt atcataaaca aaacgagata aataataaca 60 attaagcaac caaaatgcattaaaaaacac aataaaaa atg tta cct cac agt agt 116 Met Leu Pro His Ser Ser1 5 gca tta gtt tta ttt tta ttt ttt tta ttt ttc tta ttt aca cct atc 164Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu Phe Thr Pro Ile 10 15 20ttg tgc ata cta tgg gat aac cta gat cag cat ttg tgc aga gtt caa 212 LeuCys Ile Leu Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln 25 30 35 tttaac ggg atc acg gaa gga aaa ccg ttc cga tat aaa gat cat agg 260 Phe AsnGly Ile Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Arg 40 45 50 aat gatgta tat tgt tct tat ttg gga att cct tat gcc gaa ccg cct 308 Asn Asp ValTyr Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro 55 60 65 70 ttt ggacca tta cga ttt cag tct cca aaa cca ata tca aat cca aaa 356 Phe Gly ProLeu Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys 75 80 85 aca gga ttcgta cag gct cga act ttg gga gac aaa tgt ttc cag gaa 404 Thr Gly Phe ValGln Ala Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu 90 95 100 agt cta atatat tct tat gca gga agc gaa gat tgc tta tat ctg aat 452 Ser Leu Ile TyrSer Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn 105 110 115 ata ttc acgcca gag act gtt aat tct gcg aac aat aca aaa tat cct 500 Ile Phe Thr ProGlu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro 120 125 130 gta atg ttctgg atc cat gga ggc gca ttc aac caa gga tca gga tct 548 Val Met Phe TrpIle His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser 135 140 145 150 tat aatttt ttt gga cct gat tat ttg atc agg gaa gga att att ttg 596 Tyr Asn PhePhe Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu 155 160 165 gtc actatc aac tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg 644 Val Thr IleAsn Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro 170 175 180 gaa tgggat atc cat gga aat atg ggt cta aaa gac cag aga ttg gca 692 Glu Trp AspIle His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala 185 190 195 cta aaatgg gtt tac gac aac atc gaa aag ttt ggt gga gac aga gaa 740 Leu Lys TrpVal Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Glu 200 205 210 aaa attaca att gct gga gaa tct gct gga gca gca agt gtc cat ttt 788 Lys Ile ThrIle Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe 215 220 225 230 ctgatg atg gac aac tcg act aga aaa tac tac caa agg gcc att ttg 836 Leu MetMet Asp Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu 235 240 245 cagagt ggg aca tta cta aat ccg act gct aat caa att caa ctt ctg 884 Gln SerGly Thr Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Leu Leu 250 255 260 cataga ttt gaa aaa ctc aaa caa gtg cta aac atc acg caa aaa caa 932 His ArgPhe Glu Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln 265 270 275 gaactc cta aac ctg gat aaa aac cta att tta cga gca gcc tta aac 980 Glu LeuLeu Asn Leu Asp Lys Asn Leu Ile Leu Arg Ala Ala Leu Asn 280 285 290 agagtt cct gat agc aac gac cat gac cga gac aca gta cca gta ttt 1028 Arg ValPro Asp Ser Asn Asp His Asp Arg Asp Thr Val Pro Val Phe 295 300 305 310aat cca gtc tta gaa tca cca gaa tct cca gat cca ata aca ttt cca 1076 AsnPro Val Leu Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro 315 320 325tct gcc ttg gaa aga atg aga aat ggt gaa ttt cct gat gtc gat gtc 1124 SerAla Leu Glu Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val 330 335 340atc att ggt ttc aat agt gct gaa ggt tta aga tct atg gca aga gta 1172 IleIle Gly Phe Asn Ser Ala Glu Gly Leu Arg Ser Met Ala Arg Val 345 350 355acc aga gga aac atg gaa gtt cac aag act ttg aca aat ata gaa agg 1220 ThrArg Gly Asn Met Glu Val His Lys Thr Leu Thr Asn Ile Glu Arg 360 365 370gct ata cct aga gat gct aat att tgg aaa aat cca aat ggt att gag 1268 AlaIle Pro Arg Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu 375 380 385390 gag aaa aaa cta ata aaa atg ctt aca gag ttt tat gac caa gtg aaa 1316Glu Lys Lys Leu Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys 395 400405 gaa caa aac gat gac att gaa gcc tac gtc caa cta aaa ggc gat gct 1364Glu Gln Asn Asp Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala 410 415420 ggt tac ctc caa gga atc tac cgt acc ttg aaa gcc ata ttt ttc aat 1412Gly Tyr Leu Gln Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn 425 430435 gaa ttc aga agg aat tcc aat ttg tat ttg tac agg tta tca gac gat 1460Glu Phe Arg Arg Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp 440 445450 acg tat agt gta tat aaa agt tat atc ttg ccc tat cga tgg ggt tcc 1508Thr Tyr Ser Val Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser 455 460465 470 ttg cca gga gtt agt cat ggt gat gat tta gga tat ctt ttt gca aac1556 Leu Pro Gly Val Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn 475480 485 tcg ttg gat gtt cct att ttg gga aca acg cac att tct ata ccg caa1604 Ser Leu Asp Val Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln 490495 500 gat gct atg cag act ctg gaa agg atg gtc agg atc tgg acc aat ttt1652 Asp Ala Met Gln Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn Phe 505510 515 gta aag aat gga aaa cct aca tca aac act gaa gat gca tca tgt gat1700 Val Lys Asn Gly Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys Asp 520525 530 aca aaa aga cat tta aac gac att ttt tgg gaa cca tac aac gac gaa1748 Thr Lys Arg His Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp Glu 535540 545 550 gaa cca aaa tat ttg gac atg gga aaa gaa aat ttt gaa atg aaaaat 1796 Glu Pro Lys Tyr Leu Asp Met Gly Lys Glu Asn Phe Glu Met Lys Asn555 560 565 att ttg gaa cta aaa cgc atg atg ctt tgg gat gaa gtt tat agaaat 1844 Ile Leu Glu Leu Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg Asn570 575 580 gcg aat ttg cgg ttt aga gtc tgt aat gaa gaa agt att aga tga1889 Ala Asn Leu Arg Phe Arg Val Cys Asn Glu Glu Ser Ile Arg 585 590 595gtttttttaa ttttacatac agccgagagg aaacatgact aaaattggaa agaaaaatca 1949gaaaaagaaa aatcacatgg accatagtaa ctttattaca tgatttagtt tcaagtgtat 2009caagaaaact tattgcatca aagaaaatat tattttgcca aaattcttgg aaaaacactt 2069tttatgactg acatggccca taattgaagc tttttcttct tttaccaaat cgccaaattt 2129tgtagcgtca gacacattta tttatgacat ggcaattaat gtgttaaaca ttcaactcta 2189tattaaaaat ggtagtattt tctaataaga aggttatata aaaagacttg aaaataataa 2249gatttgctcg gctatatata aaaacttanc gtctcgttat gctaaacttt tttgatggta 2309aaaatatgtt gattttccta ataatctaag atattatatt ttagattaaa ttaaaatatg 2369atattttcaa ttaattaatt ttagttttaa atgtactata tttaccagta ctatgaaact 2429attttaaata tattttttat tacaatattt atttctcaaa aatgtttagt gtaacaagac 2489cattaaatta gagttaatgt tgtaaattaa actatttttt atctatcaca accgcttaat 2549tggtgcaaag aaaaatttta ctgtgataat atttgacatt tacacaatat tacgaattgt 2609aaactcacaa ttatgtgaat attgtttttt gttaaaaaaa catacatgac ttttctatat 2669cattttatat tacggtgata tggattaatg tcaacatgta aaatacaaat gcggttgtta 2729aaaataatct gtattaaaat tgttatataa aatctgaata aatgtacttt taagtaaaaa 2789aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2836 25 596 PRTCtenocephalides felis 25 Met Leu Pro His Ser Ser Ala Leu Val Leu Phe LeuPhe Phe Leu Phe 1 5 10 15 Phe Leu Phe Thr Pro Ile Leu Cys Ile Leu TrpAsp Asn Leu Asp Gln 20 25 30 His Leu Cys Arg Val Gln Phe Asn Gly Ile ThrGlu Gly Lys Pro Phe 35 40 45 Arg Tyr Lys Asp His Arg Asn Asp Val Tyr CysSer Tyr Leu Gly Ile 50 55 60 Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu ArgPhe Gln Ser Pro Lys 65 70 75 80 Pro Ile Ser Asn Pro Lys Thr Gly Phe ValGln Ala Arg Thr Leu Gly 85 90 95 Asp Lys Cys Phe Gln Glu Ser Leu Ile TyrSer Tyr Ala Gly Ser Glu 100 105 110 Asp Cys Leu Tyr Leu Asn Ile Phe ThrPro Glu Thr Val Asn Ser Ala 115 120 125 Asn Asn Thr Lys Tyr Pro Val MetPhe Trp Ile His Gly Gly Ala Phe 130 135 140 Asn Gln Gly Ser Gly Ser TyrAsn Phe Phe Gly Pro Asp Tyr Leu Ile 145 150 155 160 Arg Glu Gly Ile IleLeu Val Thr Ile Asn Tyr Arg Leu Gly Val Phe 165 170 175 Gly Phe Leu SerAla Pro Glu Trp Asp Ile His Gly Asn Met Gly Leu 180 185 190 Lys Asp GlnArg Leu Ala Leu Lys Trp Val Tyr Asp Asn Ile Glu Lys 195 200 205 Phe GlyGly Asp Arg Glu Lys Ile Thr Ile Ala Gly Glu Ser Ala Gly 210 215 220 AlaAla Ser Val His Phe Leu Met Met Asp Asn Ser Thr Arg Lys Tyr 225 230 235240 Tyr Gln Arg Ala Ile Leu Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala 245250 255 Asn Gln Ile Gln Leu Leu His Arg Phe Glu Lys Leu Lys Gln Val Leu260 265 270 Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn Leu Asp Lys Asn LeuIle 275 280 285 Leu Arg Ala Ala Leu Asn Arg Val Pro Asp Ser Asn Asp HisAsp Arg 290 295 300 Asp Thr Val Pro Val Phe Asn Pro Val Leu Glu Ser ProGlu Ser Pro 305 310 315 320 Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu ArgMet Arg Asn Gly Glu 325 330 335 Phe Pro Asp Val Asp Val Ile Ile Gly PheAsn Ser Ala Glu Gly Leu 340 345 350 Arg Ser Met Ala Arg Val Thr Arg GlyAsn Met Glu Val His Lys Thr 355 360 365 Leu Thr Asn Ile Glu Arg Ala IlePro Arg Asp Ala Asn Ile Trp Lys 370 375 380 Asn Pro Asn Gly Ile Glu GluLys Lys Leu Ile Lys Met Leu Thr Glu 385 390 395 400 Phe Tyr Asp Gln ValLys Glu Gln Asn Asp Asp Ile Glu Ala Tyr Val 405 410 415 Gln Leu Lys GlyAsp Ala Gly Tyr Leu Gln Gly Ile Tyr Arg Thr Leu 420 425 430 Lys Ala IlePhe Phe Asn Glu Phe Arg Arg Asn Ser Asn Leu Tyr Leu 435 440 445 Tyr ArgLeu Ser Asp Asp Thr Tyr Ser Val Tyr Lys Ser Tyr Ile Leu 450 455 460 ProTyr Arg Trp Gly Ser Leu Pro Gly Val Ser His Gly Asp Asp Leu 465 470 475480 Gly Tyr Leu Phe Ala Asn Ser Leu Asp Val Pro Ile Leu Gly Thr Thr 485490 495 His Ile Ser Ile Pro Gln Asp Ala Met Gln Thr Leu Glu Arg Met Val500 505 510 Arg Ile Trp Thr Asn Phe Val Lys Asn Gly Lys Pro Thr Ser AsnThr 515 520 525 Glu Asp Ala Ser Cys Asp Thr Lys Arg His Leu Asn Asp IlePhe Trp 530 535 540 Glu Pro Tyr Asn Asp Glu Glu Pro Lys Tyr Leu Asp MetGly Lys Glu 545 550 555 560 Asn Phe Glu Met Lys Asn Ile Leu Glu Leu LysArg Met Met Leu Trp 565 570 575 Asp Glu Val Tyr Arg Asn Ala Asn Leu ArgPhe Arg Val Cys Asn Glu 580 585 590 Glu Ser Ile Arg 595 26 2836 DNACtenocephalides felis misc_feature (559)..(559) n = unknown 26tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttacttaaaa 60gtacatttat tcagatttta tataacaatt ttaatacaga ttatttttaa caaccgcatt 120tgtattttac atgttgacat taatccatat caccgtaata taaaatgata tagaaaagtc 180atgtatgttt ttttaacaaa aaacaatatt cacataattg tgagtttaca attcgtaata 240ttgtgtaaat gtcaaatatt atcacagtaa aatttttctt tgcaccaatt aagcggttgt 300gatagataaa aaatagttta atttacaaca ttaactctaa tttaatggtc ttgttacact 360aaacattttt gagaaataaa tattgtaata aaaaatatat ttaaaatagt ttcatagtac 420tggtaaatat agtacattta aaactaaaat taattaattg aaaatatcat attttaattt 480aatctaaaat ataatatctt agattattag gaaaatcaac atatttttac catcaaaaaa 540gtttagcata acgagacgnt aagtttttat atatagccga gcaaatctta ttattttcaa 600gtctttttat ataaccttct tattagaaaa tactaccatt tttaatatag agttgaatgt 660ttaacacatt aattgccatg tcataaataa atgtgtctga cgctacaaaa tttggcgatt 720tggtaaaaga agaaaaagct tcaattatgg gccatgtcag tcataaaaag tgtttttcca 780agaattttgg caaaataata ttttctttga tgcaataagt tttcttgata cacttgaaac 840taaatcatgt aataaagtta ctatggtcca tgtgattttt ctttttctga tttttctttc 900caattttagt catgtttcct ctcggctgta tgtaaaatta aaaaaactca tctaatactt 960tcttcattac agactctaaa ccgcaaattc gcatttctat aaacttcatc ccaaagcatc 1020atgcgtttta gttccaaaat atttttcatt tcaaaatttt cttttcccat gtccaaatat 1080tttggttctt cgtcgttgta tggttcccaa aaaatgtcgt ttaaatgtct ttttgtatca 1140catgatgcat cttcagtgtt tgatgtaggt tttccattct ttacaaaatt ggtccagatc 1200ctgaccatcc tttccagagt ctgcatagca tcttgcggta tagaaatgtg cgttgttccc 1260aaaataggaa catccaacga gtttgcaaaa agatatccta aatcatcacc atgactaact 1320cctggcaagg aaccccatcg atagggcaag atataacttt tatatacact atacgtatcg 1380tctgataacc tgtacaaata caaattggaa ttccttctga attcattgaa aaatatggct 1440ttcaaggtac ggtagattcc ttggaggtaa ccagcatcgc cttttagttg gacgtaggct 1500tcaatgtcat cgttttgttc tttcacttgg tcataaaact ctgtaagcat ttttattagt 1560tttttctcct caataccatt tggatttttc caaatattag catctctagg tatagccctt 1620tctatatttg tcaaagtctt gtgaacttcc atgtttcctc tggttactct tgccatagat 1680cttaaacctt cagcactatt gaaaccaatg atgacatcga catcaggaaa ttcaccattt 1740ctcattcttt ccaaggcaga tggaaatgtt attggatctg gagattctgg tgattctaag 1800actggattaa atactggtac tgtgtctcgg tcatggtcgt tgctatcagg aactctgttt 1860aaggctgctc gtaaaattag gtttttatcc aggtttagga gttcttgttt ttgcgtgatg 1920tttagcactt gtttgagttt ttcaaatcta tgcagaagtt gaatttgatt agcagtcgga 1980tttagtaatg tcccactctg caaaatggcc ctttggtagt attttctagt cgagttgtcc 2040atcatcagaa aatggacact tgctgctcca gcagattctc cagcaattgt aattttttct 2100ctgtctccac caaacttttc gatgttgtcg taaacccatt ttagtgccaa tctctggtct 2160tttagaccca tatttccatg gatatcccat tccggcgctg atagaaaacc gaaaactcct 2220aatctatagt tgatagtgac caaaataatt ccttccctga tcaaataatc aggtccaaaa 2280aaattataag atcctgatcc ttggttgaat gcgcctccat ggatccagaa cattacagga 2340tattttgtat tgttcgcaga attaacagtc tctggcgtga atatattcag atataagcaa 2400tcttcgcttc ctgcataaga atatattaga ctttcctgga aacatttgtc tcccaaagtt 2460cgagcctgta cgaatcctgt ttttggattt gatattggtt ttggagactg aaatcgtaat 2520ggtccaaaag gcggttcggc ataaggaatt cccaaataag aacaatatac atcattccta 2580tgatctttat atcggaacgg ttttccttcc gtgatcccgt taaattgaac tctgcacaaa 2640tgctgatcta ggttatccca tagtatgcac aagataggtg taaataagaa aaataaaaaa 2700aataaaaata aaactaatgc actactgtga ggtaacattt tttattgtgt tttttaatgc 2760attttggttg cttaattgtt attatttatc tcgttttgtt tatgataaaa tagacgtttt 2820gaagacgaca tgtcta 2836 27 1710 DNA Ctenocephalides felis exon(1)..(1710) 27 tgg gat aac cta gat cag cat ttg tgc aga gtt caa ttt aacggg atc 48 Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln Phe Asn GlyIle 1 5 10 15 acg gaa gga aaa ccg ttc cga tat aaa gat cat agg aat gatgta tat 96 Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Arg Asn Asp ValTyr 20 25 30 tgt tct tat ttg gga att cct tat gcc gaa ccg cct ttt gga ccatta 144 Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu35 40 45 cga ttt cag tct cca aaa cca ata tca aat cca aaa aca gga ttc gta192 Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 5055 60 cag gct cga act ttg gga gac aaa tgt ttc cag gaa agt cta ata tat240 Gln Ala Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr 6570 75 80 tct tat gca gga agc gaa gat tgc tta tat ctg aat ata ttc acg cca288 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro 8590 95 gag act gtt aat tct gcg aac aat aca aaa tat cct gta atg ttc tgg336 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val Met Phe Trp 100105 110 atc cat gga ggc gca ttc aac caa gga tca gga tct tat aat ttt ttt384 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe 115120 125 gga cct gat tat ttg atc agg gaa gga att att ttg gtc act atc aac432 Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val Thr Ile Asn 130135 140 tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg gaa tgg gat atc480 Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile 145150 155 160 cat gga aat atg ggt cta aaa gac cag aga ttg gca cta aaa tgggtt 528 His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp Val165 170 175 tac gac aac atc gaa aag ttt ggt gga gac aga gaa aaa att acaatt 576 Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Glu Lys Ile Thr Ile180 185 190 gct gga gaa tct gct gga gca gca agt gtc cat ttt ctg atg atggac 624 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp195 200 205 aac tcg act aga aaa tac tac caa agg gcc att ttg cag agt gggaca 672 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln Ser Gly Thr210 215 220 tta cta aat ccg act gct aat caa att caa ctt ctg cat aga tttgaa 720 Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Leu Leu His Arg Phe Glu225 230 235 240 aaa ctc aaa caa gtg cta aac atc acg caa aaa caa gaa ctccta aac 768 Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln Glu Leu LeuAsn 245 250 255 ctg gat aaa aac cta att tta cga gca gcc tta aac aga gttcct gat 816 Leu Asp Lys Asn Leu Ile Leu Arg Ala Ala Leu Asn Arg Val ProAsp 260 265 270 agc aac gac cat gac cga gac aca gta cca gta ttt aat ccagtc tta 864 Ser Asn Asp His Asp Arg Asp Thr Val Pro Val Phe Asn Pro ValLeu 275 280 285 gaa tca cca gaa tct cca gat cca ata aca ttt cca tct gccttg gaa 912 Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala LeuGlu 290 295 300 aga atg aga aat ggt gaa ttt cct gat gtc gat gtc atc attggt ttc 960 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile GlyPhe 305 310 315 320 aat agt gct gaa ggt tta aga tct atg gca aga gta accaga gga aac 1008 Asn Ser Ala Glu Gly Leu Arg Ser Met Ala Arg Val Thr ArgGly Asn 325 330 335 atg gaa gtt cac aag act ttg aca aat ata gaa agg gctata cct aga 1056 Met Glu Val His Lys Thr Leu Thr Asn Ile Glu Arg Ala IlePro Arg 340 345 350 gat gct aat att tgg aaa aat cca aat ggt att gag gagaaa aaa cta 1104 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu Glu LysLys Leu 355 360 365 ata aaa atg ctt aca gag ttt tat gac caa gtg aaa gaacaa aac gat 1152 Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys Glu GlnAsn Asp 370 375 380 gac att gaa gcc tac gtc caa cta aaa ggc gat gct ggttac ctc caa 1200 Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala Gly TyrLeu Gln 385 390 395 400 gga atc tac cgt acc ttg aaa gcc ata ttt ttc aatgaa ttc aga agg 1248 Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn GluPhe Arg Arg 405 410 415 aat tcc aat ttg tat ttg tac agg tta tca gac gatacg tat agt gta 1296 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp ThrTyr Ser Val 420 425 430 tat aaa agt tat atc ttg ccc tat cga tgg ggt tccttg cca gga gtt 1344 Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser LeuPro Gly Val 435 440 445 agt cat ggt gat gat tta gga tat ctt ttt gca aactcg ttg gat gtt 1392 Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn SerLeu Asp Val 450 455 460 cct att ttg gga aca acg cac att tct ata ccg caagat gct atg cag 1440 Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln AspAla Met Gln 465 470 475 480 act ctg gaa agg atg gtc agg atc tgg acc aatttt gta aag aat gga 1488 Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn PheVal Lys Asn Gly 485 490 495 aaa cct aca tca aac act gaa gat gca tca tgtgat aca aaa aga cat 1536 Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys AspThr Lys Arg His 500 505 510 tta aac gac att ttt tgg gaa cca tac aac gacgaa gaa cca aaa tat 1584 Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp GluGlu Pro Lys Tyr 515 520 525 ttg gac atg gga aaa gaa aat ttt gaa atg aaaaat att ttg gaa cta 1632 Leu Asp Met Gly Lys Glu Asn Phe Glu Met Lys AsnIle Leu Glu Leu 530 535 540 aaa cgc atg atg ctt tgg gat gaa gtt tat agaaat gcg aat ttg cgg 1680 Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg AsnAla Asn Leu Arg 545 550 555 560 ttt aga gtc tgt aat gaa gaa agt att aga1710 Phe Arg Val Cys Asn Glu Glu Ser Ile Arg 565 570 28 1788 DNACtenocephalides felis exon (1)..(1788) 28 atg tta cct cac agt agt gcatta gtt tta ttt tta ttt ttt tta ttt 48 Met Leu Pro His Ser Ser Ala LeuVal Leu Phe Leu Phe Phe Leu Phe 1 5 10 15 ttc tta ttt aca cct atc ttgtgc ata cta tgg gat aac cta gat cag 96 Phe Leu Phe Thr Pro Ile Leu CysIle Leu Trp Asp Asn Leu Asp Gln 20 25 30 cat ttg tgc aga gtt caa ttt aacggg atc acg gaa gga aaa ccg ttc 144 His Leu Cys Arg Val Gln Phe Asn GlyIle Thr Glu Gly Lys Pro Phe 35 40 45 cga tat aaa gat cat agg aat gat gtatat tgt tct tat ttg gga att 192 Arg Tyr Lys Asp His Arg Asn Asp Val TyrCys Ser Tyr Leu Gly Ile 50 55 60 cct tat gcc gaa ccg cct ttt gga cca ttacga ttt cag tct cca aaa 240 Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu ArgPhe Gln Ser Pro Lys 65 70 75 80 cca ata tca aat cca aaa aca gga ttc gtacag gct cga act ttg gga 288 Pro Ile Ser Asn Pro Lys Thr Gly Phe Val GlnAla Arg Thr Leu Gly 85 90 95 gac aaa tgt ttc cag gaa agt cta ata tat tcttat gca gga agc gaa 336 Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr Ser TyrAla Gly Ser Glu 100 105 110 gat tgc tta tat ctg aat ata ttc acg cca gagact gtt aat tct gcg 384 Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro Glu ThrVal Asn Ser Ala 115 120 125 aac aat aca aaa tat cct gta atg ttc tgg atccat gga ggc gca ttc 432 Asn Asn Thr Lys Tyr Pro Val Met Phe Trp Ile HisGly Gly Ala Phe 130 135 140 aac caa gga tca gga tct tat aat ttt ttt ggacct gat tat ttg atc 480 Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly ProAsp Tyr Leu Ile 145 150 155 160 agg gaa gga att att ttg gtc act atc aactat aga tta gga gtt ttc 528 Arg Glu Gly Ile Ile Leu Val Thr Ile Asn TyrArg Leu Gly Val Phe 165 170 175 ggt ttt cta tca gcg ccg gaa tgg gat atccat gga aat atg ggt cta 576 Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile HisGly Asn Met Gly Leu 180 185 190 aaa gac cag aga ttg gca cta aaa tgg gtttac gac aac atc gaa aag 624 Lys Asp Gln Arg Leu Ala Leu Lys Trp Val TyrAsp Asn Ile Glu Lys 195 200 205 ttt ggt gga gac aga gaa aaa att aca attgct gga gaa tct gct gga 672 Phe Gly Gly Asp Arg Glu Lys Ile Thr Ile AlaGly Glu Ser Ala Gly 210 215 220 gca gca agt gtc cat ttt ctg atg atg gacaac tcg act aga aaa tac 720 Ala Ala Ser Val His Phe Leu Met Met Asp AsnSer Thr Arg Lys Tyr 225 230 235 240 tac caa agg gcc att ttg cag agt gggaca tta cta aat ccg act gct 768 Tyr Gln Arg Ala Ile Leu Gln Ser Gly ThrLeu Leu Asn Pro Thr Ala 245 250 255 aat caa att caa ctt ctg cat aga tttgaa aaa ctc aaa caa gtg cta 816 Asn Gln Ile Gln Leu Leu His Arg Phe GluLys Leu Lys Gln Val Leu 260 265 270 aac atc acg caa aaa caa gaa ctc ctaaac ctg gat aaa aac cta att 864 Asn Ile Thr Gln Lys Gln Glu Leu Leu AsnLeu Asp Lys Asn Leu Ile 275 280 285 tta cga gca gcc tta aac aga gtt cctgat agc aac gac cat gac cga 912 Leu Arg Ala Ala Leu Asn Arg Val Pro AspSer Asn Asp His Asp Arg 290 295 300 gac aca gta cca gta ttt aat cca gtctta gaa tca cca gaa tct cca 960 Asp Thr Val Pro Val Phe Asn Pro Val LeuGlu Ser Pro Glu Ser Pro 305 310 315 320 gat cca ata aca ttt cca tct gccttg gaa aga atg aga aat ggt gaa 1008 Asp Pro Ile Thr Phe Pro Ser Ala LeuGlu Arg Met Arg Asn Gly Glu 325 330 335 ttt cct gat gtc gat gtc atc attggt ttc aat agt gct gaa ggt tta 1056 Phe Pro Asp Val Asp Val Ile Ile GlyPhe Asn Ser Ala Glu Gly Leu 340 345 350 aga tct atg gca aga gta acc agagga aac atg gaa gtt cac aag act 1104 Arg Ser Met Ala Arg Val Thr Arg GlyAsn Met Glu Val His Lys Thr 355 360 365 ttg aca aat ata gaa agg gct atacct aga gat gct aat att tgg aaa 1152 Leu Thr Asn Ile Glu Arg Ala Ile ProArg Asp Ala Asn Ile Trp Lys 370 375 380 aat cca aat ggt att gag gag aaaaaa cta ata aaa atg ctt aca gag 1200 Asn Pro Asn Gly Ile Glu Glu Lys LysLeu Ile Lys Met Leu Thr Glu 385 390 395 400 ttt tat gac caa gtg aaa gaacaa aac gat gac att gaa gcc tac gtc 1248 Phe Tyr Asp Gln Val Lys Glu GlnAsn Asp Asp Ile Glu Ala Tyr Val 405 410 415 caa cta aaa ggc gat gct ggttac ctc caa gga atc tac cgt acc ttg 1296 Gln Leu Lys Gly Asp Ala Gly TyrLeu Gln Gly Ile Tyr Arg Thr Leu 420 425 430 aaa gcc ata ttt ttc aat gaattc aga agg aat tcc aat ttg tat ttg 1344 Lys Ala Ile Phe Phe Asn Glu PheArg Arg Asn Ser Asn Leu Tyr Leu 435 440 445 tac agg tta tca gac gat acgtat agt gta tat aaa agt tat atc ttg 1392 Tyr Arg Leu Ser Asp Asp Thr TyrSer Val Tyr Lys Ser Tyr Ile Leu 450 455 460 ccc tat cga tgg ggt tcc ttgcca gga gtt agt cat ggt gat gat tta 1440 Pro Tyr Arg Trp Gly Ser Leu ProGly Val Ser His Gly Asp Asp Leu 465 470 475 480 gga tat ctt ttt gca aactcg ttg gat gtt cct att ttg gga aca acg 1488 Gly Tyr Leu Phe Ala Asn SerLeu Asp Val Pro Ile Leu Gly Thr Thr 485 490 495 cac att tct ata ccg caagat gct atg cag act ctg gaa agg atg gtc 1536 His Ile Ser Ile Pro Gln AspAla Met Gln Thr Leu Glu Arg Met Val 500 505 510 agg atc tgg acc aat tttgta aag aat gga aaa cct aca tca aac act 1584 Arg Ile Trp Thr Asn Phe ValLys Asn Gly Lys Pro Thr Ser Asn Thr 515 520 525 gaa gat gca tca tgt gataca aaa aga cat tta aac gac att ttt tgg 1632 Glu Asp Ala Ser Cys Asp ThrLys Arg His Leu Asn Asp Ile Phe Trp 530 535 540 gaa cca tac aac gac gaagaa cca aaa tat ttg gac atg gga aaa gaa 1680 Glu Pro Tyr Asn Asp Glu GluPro Lys Tyr Leu Asp Met Gly Lys Glu 545 550 555 560 aat ttt gaa atg aaaaat att ttg gaa cta aaa cgc atg atg ctt tgg 1728 Asn Phe Glu Met Lys AsnIle Leu Glu Leu Lys Arg Met Met Leu Trp 565 570 575 gat gaa gtt tat agaaat gcg aat ttg cgg ttt aga gtc tgt aat gaa 1776 Asp Glu Val Tyr Arg AsnAla Asn Leu Arg Phe Arg Val Cys Asn Glu 580 585 590 gaa agt att aga 1788Glu Ser Ile Arg 595 29 1788 DNA Ctenocephalides felis 29 tctaatactttcttcattac agactctaaa ccgcaaattc gcatttctat aaacttcatc 60 ccaaagcatcatgcgtttta gttccaaaat atttttcatt tcaaaatttt cttttcccat 120 gtccaaatattttggttctt cgtcgttgta tggttcccaa aaaatgtcgt ttaaatgtct 180 ttttgtatcacatgatgcat cttcagtgtt tgatgtaggt tttccattct ttacaaaatt 240 ggtccagatcctgaccatcc tttccagagt ctgcatagca tcttgcggta tagaaatgtg 300 cgttgttcccaaaataggaa catccaacga gtttgcaaaa agatatccta aatcatcacc 360 atgactaactcctggcaagg aaccccatcg atagggcaag atataacttt tatatacact 420 atacgtatcgtctgataacc tgtacaaata caaattggaa ttccttctga attcattgaa 480 aaatatggctttcaaggtac ggtagattcc ttggaggtaa ccagcatcgc cttttagttg 540 gacgtaggcttcaatgtcat cgttttgttc tttcacttgg tcataaaact ctgtaagcat 600 ttttattagttttttctcct caataccatt tggatttttc caaatattag catctctagg 660 tatagccctttctatatttg tcaaagtctt gtgaacttcc atgtttcctc tggttactct 720 tgccatagatcttaaacctt cagcactatt gaaaccaatg atgacatcga catcaggaaa 780 ttcaccatttctcattcttt ccaaggcaga tggaaatgtt attggatctg gagattctgg 840 tgattctaagactggattaa atactggtac tgtgtctcgg tcatggtcgt tgctatcagg 900 aactctgtttaaggctgctc gtaaaattag gtttttatcc aggtttagga gttcttgttt 960 ttgcgtgatgtttagcactt gtttgagttt ttcaaatcta tgcagaagtt gaatttgatt 1020 agcagtcggatttagtaatg tcccactctg caaaatggcc ctttggtagt attttctagt 1080 cgagttgtccatcatcagaa aatggacact tgctgctcca gcagattctc cagcaattgt 1140 aattttttctctgtctccac caaacttttc gatgttgtcg taaacccatt ttagtgccaa 1200 tctctggtcttttagaccca tatttccatg gatatcccat tccggcgctg atagaaaacc 1260 gaaaactcctaatctatagt tgatagtgac caaaataatt ccttccctga tcaaataatc 1320 aggtccaaaaaaattataag atcctgatcc ttggttgaat gcgcctccat ggatccagaa 1380 cattacaggatattttgtat tgttcgcaga attaacagtc tctggcgtga atatattcag 1440 atataagcaatcttcgcttc ctgcataaga atatattaga ctttcctgga aacatttgtc 1500 tcccaaagttcgagcctgta cgaatcctgt ttttggattt gatattggtt ttggagactg 1560 aaatcgtaatggtccaaaag gcggttcggc ataaggaatt cccaaataag aacaatatac 1620 atcattcctatgatctttat atcggaacgg ttttccttcc gtgatcccgt taaattgaac 1680 tctgcacaaatgctgatcta ggttatccca tagtatgcac aagataggtg taaataagaa 1740 aaataaaaaaaataaaaata aaactaatgc actactgtga ggtaacat 1788 30 2801 DNACtenocephalides felis CDS (99)..(1886) 30 gacatgtcgt cttcaaaacgtctattttat cataaacaaa acgagataaa taataacaat 60 taagcatcca aaatgcattaaaaaaaacat cataaaaa atg tta cct cac agt gca 116 Met Leu Pro His Ser Ala1 5 tta gtt tta ttt tta ttt ttt tta ttt ttc tta ttt aca cct gtc ttg 164Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu Phe Thr Pro Val Leu 10 15 20tgc ata cta tgg gat aac cta gat cag cat ttg tgc aga gtt caa ttt 212 CysIle Leu Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln Phe 25 30 35 aacggg atc acg gaa gga aaa ccg ttc cga tat aaa gat cat aaa aat 260 Asn GlyIle Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Lys Asn 40 45 50 gat gtatat tgt tcc tat ttg gga att cct tat gca gaa ccg cct att 308 Asp Val TyrCys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Ile 55 60 65 70 gga ccattg cga ttt cag tct cca aaa cca ata tca aat cca aaa aca 356 Gly Pro LeuArg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr 75 80 85 gga ttc gttcag gct cgg tct tta gga gac aaa tgt ttc cag gaa agt 404 Gly Phe Val GlnAla Arg Ser Leu Gly Asp Lys Cys Phe Gln Glu Ser 90 95 100 cta ata tattct tat gca gga agc gaa gat tgc tta tat ctg aat ata 452 Leu Ile Tyr SerTyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile 105 110 115 ttc acg ccagag act gtt aat tct gcg aac aat aca aaa tat cct gta 500 Phe Thr Pro GluThr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val 120 125 130 atg ttc tggatc cat gga ggc gca ttc aac caa gga tca gga tct tat 548 Met Phe Trp IleHis Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr 135 140 145 150 aat tttttt gga cct gat tat ttg atc agg gaa gga att att ttg gtc 596 Asn Phe PheGly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val 155 160 165 act atcaac tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg gaa 644 Thr Ile AsnTyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu 170 175 180 tgg gatatc cat gga aat atg ggt cta aaa gac cag aga ttg gca cta 692 Trp Asp IleHis Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu 185 190 195 aaa tgggtt tat gac aac atc gaa aaa ttt ggt gga gac aga gat aaa 740 Lys Trp ValTyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Asp Lys 200 205 210 atc actata gct gga gaa tct gct gga gca gca agt gtt cat ttt ctg 788 Ile Thr IleAla Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu 215 220 225 230 atgatg gac aat tct act aga aaa tac tac caa agg gca att ttg cag 836 Met MetAsp Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln 235 240 245 agtggg aca tta ctc aat ccg act gct aat caa att caa cct ctg cat 884 Ser GlyThr Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Pro Leu His 250 255 260 agattt gaa aaa cta aaa caa gtg ctg aac atc acg caa aaa caa gaa 932 Arg PheGlu Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln Glu 265 270 275 ctccta aat ctg gac aaa aat caa att ttg cga gca gcc tta aac aga 980 Leu LeuAsn Leu Asp Lys Asn Gln Ile Leu Arg Ala Ala Leu Asn Arg 280 285 290 gtccca gat aac aac gac cac gaa agg gac aca gta cca gta ttt aat 1028 Val ProAsp Asn Asn Asp His Glu Arg Asp Thr Val Pro Val Phe Asn 295 300 305 310cca gtc cta gaa tca cca gaa tct cca gac cca ata aca ttt cca tct 1076 ProVal Leu Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser 315 320 325gct tta gaa aga atg aga aat ggt gaa ttt cct gac gtt gat gtc atc 1124 AlaLeu Glu Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile 330 335 340att gga ttc aat agt gct gaa ggt tta aga tct atg cca aga gta acc 1172 IleGly Phe Asn Ser Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr 345 350 355aga gga aac atg gaa gtt tac aag act ttg aca aat ata gag aga gct 1220 ArgGly Asn Met Glu Val Tyr Lys Thr Leu Thr Asn Ile Glu Arg Ala 360 365 370ata cct aga gat gct aat att tgg aaa aat cct aat ggc att gag gag 1268 IlePro Arg Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu Glu 375 380 385390 aaa aaa ctt ata aaa atg ctt aca gag ttt tat gac caa gtt aaa gaa 1316Lys Lys Leu Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys Glu 395 400405 caa aac gat gac atc gaa gcc tat gtc caa cta aaa ggc gat gct ggt 1364Gln Asn Asp Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala Gly 410 415420 tat ctc caa gga att tac cgt acc ttg aaa gcc ata ttt ttc aat gaa 1412Tyr Leu Gln Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn Glu 425 430435 atc aaa aga aat tcc aac ttg tat ttg tat agg tta tca gat gat acg 1460Ile Lys Arg Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp Thr 440 445450 tat agt gta tat aaa agt tat atc ttg ccc tat cga tgg ggt tcc ttg 1508Tyr Ser Val Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser Leu 455 460465 470 cca gga gtt agt cat ggt gat gat tta gga tat ctt ttt gca aac tct1556 Pro Gly Val Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn Ser 475480 485 ttg gat gtt cct att ttg gga aca acg cac att tct ata ccg caa gat1604 Leu Asp Val Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln Asp 490495 500 gct atg cag act ctg gaa agg atg gtc agg atc tgg acc aat ttt gta1652 Ala Met Gln Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn Phe Val 505510 515 aag aat gga aaa cct aca tca aac act gaa gat gca tca tgt gat aca1700 Lys Asn Gly Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys Asp Thr 520525 530 aaa aga cat tta aac gac att ttt tgg gaa cca tac aac gac gaa gaa1748 Lys Arg His Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp Glu Glu 535540 545 550 cca aaa tat ttg gac atg gga aaa gaa cat ttt gaa atg aaa aatatt 1796 Pro Lys Tyr Leu Asp Met Gly Lys Glu His Phe Glu Met Lys Asn Ile555 560 565 ttg gaa cta aaa cgc atg atg ctt tgg gat gaa gtt tat aga aatgcg 1844 Leu Glu Leu Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg Asn Ala570 575 580 aat ttg cgg ttt aga gtc tgt aat gaa gaa agt att aga tga 1886Asn Leu Arg Phe Arg Val Cys Asn Glu Glu Ser Ile Arg 585 590 595gtttttttaa ttttacatac agccgagagg aaacatgact aaaattggaa agaaaaatca 1946gaaaaagaaa aatcacatgg accatagtaa ctttattaca tgatttagtt tcaagtgtat 2006caagaaaact tattgcatca aagaaaatat tattttgcca aaattcttgg aaaaacactt 2066tttatgactg acatggccca taattgaagc tttttcttct tttaccaaat cgccaaattt 2126tgtagcgtca gacacattta tttatgacat ggcaattaat gtgttaaaca ttcaactcta 2186tattaaaaat ggtagtattt tctaataaga aggttatata aaaagacttg aaaataataa 2246gatttgctcg gctatatata aaaacttanc gtctcgttat gctaaacttt tttgatggta 2306aaaatatgtt gattttccta ataatctaag atattatatt ttagattaaa ttaaaatatg 2366atattttcaa ttaattaatt ttagttttaa atgtactata tttaccagta ctatgaaact 2426attttaaata tattttttat tacaatattt atttctcaaa aatgtttagt gtaacaagac 2486cattaaatta gagttaatgt tgtaaattaa actatttttt atctatcaca accgcttaat 2546tggtgcaaag aaaaatttta ctgtgataat atttgacatt tacacaatat tacgaattgt 2606aaactcacaa ttatgtgaat attgtttttt gttaaaaaaa catacatgac ttttctatat 2666cattttatat tacggtgata tggattaatg tcaacatgta aaatacaaat gcggttgtta 2726aaaataatct gtattaaaat tgttatataa aatctgaata aatgtacttt taagtaaaaa 2786aaaaaaaaaa aaaaa 2801 31 595 PRT Ctenocephalides felis 31 Met Leu ProHis Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe 1 5 10 15 Leu PheThr Pro Val Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln His 20 25 30 Leu CysArg Val Gln Phe Asn Gly Ile Thr Glu Gly Lys Pro Phe Arg 35 40 45 Tyr LysAsp His Lys Asn Asp Val Tyr Cys Ser Tyr Leu Gly Ile Pro 50 55 60 Tyr AlaGlu Pro Pro Ile Gly Pro Leu Arg Phe Gln Ser Pro Lys Pro 65 70 75 80 IleSer Asn Pro Lys Thr Gly Phe Val Gln Ala Arg Ser Leu Gly Asp 85 90 95 LysCys Phe Gln Glu Ser Leu Ile Tyr Ser Tyr Ala Gly Ser Glu Asp 100 105 110Cys Leu Tyr Leu Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala Asn 115 120125 Asn Thr Lys Tyr Pro Val Met Phe Trp Ile His Gly Gly Ala Phe Asn 130135 140 Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro Asp Tyr Leu Ile Arg145 150 155 160 Glu Gly Ile Ile Leu Val Thr Ile Asn Tyr Arg Leu Gly ValPhe Gly 165 170 175 Phe Leu Ser Ala Pro Glu Trp Asp Ile His Gly Asn MetGly Leu Lys 180 185 190 Asp Gln Arg Leu Ala Leu Lys Trp Val Tyr Asp AsnIle Glu Lys Phe 195 200 205 Gly Gly Asp Arg Asp Lys Ile Thr Ile Ala GlyGlu Ser Ala Gly Ala 210 215 220 Ala Ser Val His Phe Leu Met Met Asp AsnSer Thr Arg Lys Tyr Tyr 225 230 235 240 Gln Arg Ala Ile Leu Gln Ser GlyThr Leu Leu Asn Pro Thr Ala Asn 245 250 255 Gln Ile Gln Pro Leu His ArgPhe Glu Lys Leu Lys Gln Val Leu Asn 260 265 270 Ile Thr Gln Lys Gln GluLeu Leu Asn Leu Asp Lys Asn Gln Ile Leu 275 280 285 Arg Ala Ala Leu AsnArg Val Pro Asp Asn Asn Asp His Glu Arg Asp 290 295 300 Thr Val Pro ValPhe Asn Pro Val Leu Glu Ser Pro Glu Ser Pro Asp 305 310 315 320 Pro IleThr Phe Pro Ser Ala Leu Glu Arg Met Arg Asn Gly Glu Phe 325 330 335 ProAsp Val Asp Val Ile Ile Gly Phe Asn Ser Ala Glu Gly Leu Arg 340 345 350Ser Met Pro Arg Val Thr Arg Gly Asn Met Glu Val Tyr Lys Thr Leu 355 360365 Thr Asn Ile Glu Arg Ala Ile Pro Arg Asp Ala Asn Ile Trp Lys Asn 370375 380 Pro Asn Gly Ile Glu Glu Lys Lys Leu Ile Lys Met Leu Thr Glu Phe385 390 395 400 Tyr Asp Gln Val Lys Glu Gln Asn Asp Asp Ile Glu Ala TyrVal Gln 405 410 415 Leu Lys Gly Asp Ala Gly Tyr Leu Gln Gly Ile Tyr ArgThr Leu Lys 420 425 430 Ala Ile Phe Phe Asn Glu Ile Lys Arg Asn Ser AsnLeu Tyr Leu Tyr 435 440 445 Arg Leu Ser Asp Asp Thr Tyr Ser Val Tyr LysSer Tyr Ile Leu Pro 450 455 460 Tyr Arg Trp Gly Ser Leu Pro Gly Val SerHis Gly Asp Asp Leu Gly 465 470 475 480 Tyr Leu Phe Ala Asn Ser Leu AspVal Pro Ile Leu Gly Thr Thr His 485 490 495 Ile Ser Ile Pro Gln Asp AlaMet Gln Thr Leu Glu Arg Met Val Arg 500 505 510 Ile Trp Thr Asn Phe ValLys Asn Gly Lys Pro Thr Ser Asn Thr Glu 515 520 525 Asp Ala Ser Cys AspThr Lys Arg His Leu Asn Asp Ile Phe Trp Glu 530 535 540 Pro Tyr Asn AspGlu Glu Pro Lys Tyr Leu Asp Met Gly Lys Glu His 545 550 555 560 Phe GluMet Lys Asn Ile Leu Glu Leu Lys Arg Met Met Leu Trp Asp 565 570 575 GluVal Tyr Arg Asn Ala Asn Leu Arg Phe Arg Val Cys Asn Glu Glu 580 585 590Ser Ile Arg 595 32 2801 DNA Ctenocephalides felis misc_feature(527)..(527) n = unknown at position 527 32 tttttttttt ttttttttttacttaaaagt acatttattc agattttata taacaatttt 60 aatacagatt atttttaacaaccgcatttg tattttacat gttgacatta atccatatca 120 ccgtaatata aaatgatatagaaaagtcat gtatgttttt ttaacaaaaa acaatattca 180 cataattgtg agtttacaattcgtaatatt gtgtaaatgt caaatattat cacagtaaaa 240 tttttctttg caccaattaagcggttgtga tagataaaaa atagtttaat ttacaacatt 300 aactctaatt taatggtcttgttacactaa acatttttga gaaataaata ttgtaataaa 360 aaatatattt aaaatagtttcatagtactg gtaaatatag tacatttaaa actaaaatta 420 attaattgaa aatatcatattttaatttaa tctaaaatat aatatcttag attattagga 480 aaatcaacat atttttaccatcaaaaaagt ttagcataac gagacgntaa gtttttatat 540 atagccgagc aaatcttattattttcaagt ctttttatat aaccttctta ttagaaaata 600 ctaccatttt taatatagagttgaatgttt aacacattaa ttgccatgtc ataaataaat 660 gtgtctgacg ctacaaaatttggcgatttg gtaaaagaag aaaaagcttc aattatgggc 720 catgtcagtc ataaaaagtgtttttccaag aattttggca aaataatatt ttctttgatg 780 caataagttt tcttgatacacttgaaacta aatcatgtaa taaagttact atggtccatg 840 tgatttttct ttttctgatttttctttcca attttagtca tgtttcctct cggctgtatg 900 taaaattaaa aaaactcatctaatactttc ttcattacag actctaaacc gcaaattcgc 960 atttctataa acttcatcccaaagcatcat gcgttttagt tccaaaatat ttttcatttc 1020 aaaatgttct tttcccatgtccaaatattt tggttcttcg tcgttgtatg gttcccaaaa 1080 aatgtcgttt aaatgtctttttgtatcaca tgatgcatct tcagtgtttg atgtaggttt 1140 tccattcttt acaaaattggtccagatcct gaccatcctt tccagagtct gcatagcatc 1200 ttgcggtata gaaatgtgcgttgttcccaa aataggaaca tccaaagagt ttgcaaaaag 1260 atatcctaaa tcatcaccatgactaactcc tggcaaggaa ccccatcgat agggcaagat 1320 ataactttta tatacactatacgtatcatc tgataaccta tacaaataca agttggaatt 1380 tcttttgatt tcattgaaaaatatggcttt caaggtacgg taaattcctt ggagataacc 1440 agcatcgcct tttagttggacataggcttc gatgtcatcg ttttgttctt taacttggtc 1500 ataaaactct gtaagcatttttataagttt tttctcctca atgccattag gatttttcca 1560 aatattagca tctctaggtatagctctctc tatatttgtc aaagtcttgt aaacttccat 1620 gtttcctctg gttactcttggcatagatct taaaccttca gcactattga atccaatgat 1680 gacatcaacg tcaggaaattcaccatttct cattctttct aaagcagatg gaaatgttat 1740 tgggtctgga gattctggtgattctaggac tggattaaat actggtactg tgtccctttc 1800 gtggtcgttg ttatctgggactctgtttaa ggctgctcgc aaaatttgat ttttgtccag 1860 atttaggagt tcttgtttttgcgtgatgtt cagcacttgt tttagttttt caaatctatg 1920 cagaggttga atttgattagcagtcggatt gagtaatgtc ccactctgca aaattgccct 1980 ttggtagtat tttctagtagaattgtccat catcagaaaa tgaacacttg ctgctccagc 2040 agattctcca gctatagtgattttatctct gtctccacca aatttttcga tgttgtcata 2100 aacccatttt agtgccaatctctggtcttt tagacccata tttccatgga tatcccattc 2160 cggcgctgat agaaaaccgaaaactcctaa tctatagttg atagtgacca aaataattcc 2220 ttccctgatc aaataatcaggtccaaaaaa attataagat cctgatcctt ggttgaatgc 2280 gcctccatgg atccagaacattacaggata ttttgtattg ttcgcagaat taacagtctc 2340 tggcgtgaat atattcagatataagcaatc ttcgcttcct gcataagaat atattagact 2400 ttcctggaaa catttgtctcctaaagaccg agcctgaacg aatcctgttt ttggatttga 2460 tattggtttt ggagactgaaatcgcaatgg tccaataggc ggttctgcat aaggaattcc 2520 caaataggaa caatatacatcatttttatg atctttatat cggaacggtt ttccttccgt 2580 gatcccgtta aattgaactctgcacaaatg ctgatctagg ttatcccata gtatgcacaa 2640 gacaggtgta aataagaaaaataaaaaaaa taaaaataaa actaatgcac tgtgaggtaa 2700 cattttttat gatgttttttttaatgcatt ttggatgctt aattgttatt atttatctcg 2760 ttttgtttat gataaaatagacgttttgaa gacgacatgt c 2801 33 1710 DNA Ctenocephalides felis exon(1)..(1710) 33 tgg gat aac cta gat cag cat ttg tgc aga gtt caa ttt aacggg atc 48 Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln Phe Asn GlyIle 1 5 10 15 acg gaa gga aaa ccg ttc cga tat aaa gat cat aaa aat gatgta tat 96 Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Lys Asn Asp ValTyr 20 25 30 tgt tcc tat ttg gga att cct tat gca gaa ccg cct att gga ccattg 144 Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Ile Gly Pro Leu35 40 45 cga ttt cag tct cca aaa cca ata tca aat cca aaa aca gga ttc gtt192 Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 5055 60 cag gct cgg tct tta gga gac aaa tgt ttc cag gaa agt cta ata tat240 Gln Ala Arg Ser Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr 6570 75 80 tct tat gca gga agc gaa gat tgc tta tat ctg aat ata ttc acg cca288 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro 8590 95 gag act gtt aat tct gcg aac aat aca aaa tat cct gta atg ttc tgg336 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val Met Phe Trp 100105 110 atc cat gga ggc gca ttc aac caa gga tca gga tct tat aat ttt ttt384 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe 115120 125 gga cct gat tat ttg atc agg gaa gga att att ttg gtc act atc aac432 Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val Thr Ile Asn 130135 140 tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg gaa tgg gat atc480 Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile 145150 155 160 cat gga aat atg ggt cta aaa gac cag aga ttg gca cta aaa tgggtt 528 His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp Val165 170 175 tat gac aac atc gaa aaa ttt ggt gga gac aga gat aaa atc actata 576 Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Asp Lys Ile Thr Ile180 185 190 gct gga gaa tct gct gga gca gca agt gtt cat ttt ctg atg atggac 624 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp195 200 205 aat tct act aga aaa tac tac caa agg gca att ttg cag agt gggaca 672 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln Ser Gly Thr210 215 220 tta ctc aat ccg act gct aat caa att caa cct ctg cat aga tttgaa 720 Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Pro Leu His Arg Phe Glu225 230 235 240 aaa cta aaa caa gtg ctg aac atc acg caa aaa caa gaa ctccta aat 768 Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln Glu Leu LeuAsn 245 250 255 ctg gac aaa aat caa att ttg cga gca gcc tta aac aga gtccca gat 816 Leu Asp Lys Asn Gln Ile Leu Arg Ala Ala Leu Asn Arg Val ProAsp 260 265 270 aac aac gac cac gaa agg gac aca gta cca gta ttt aat ccagtc cta 864 Asn Asn Asp His Glu Arg Asp Thr Val Pro Val Phe Asn Pro ValLeu 275 280 285 gaa tca cca gaa tct cca gac cca ata aca ttt cca tct gcttta gaa 912 Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala LeuGlu 290 295 300 aga atg aga aat ggt gaa ttt cct gac gtt gat gtc atc attgga ttc 960 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile GlyPhe 305 310 315 320 aat agt gct gaa ggt tta aga tct atg cca aga gta accaga gga aac 1008 Asn Ser Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr ArgGly Asn 325 330 335 atg gaa gtt tac aag act ttg aca aat ata gag aga gctata cct aga 1056 Met Glu Val Tyr Lys Thr Leu Thr Asn Ile Glu Arg Ala IlePro Arg 340 345 350 gat gct aat att tgg aaa aat cct aat ggc att gag gagaaa aaa ctt 1104 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu Glu LysLys Leu 355 360 365 ata aaa atg ctt aca gag ttt tat gac caa gtt aaa gaacaa aac gat 1152 Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys Glu GlnAsn Asp 370 375 380 gac atc gaa gcc tat gtc caa cta aaa ggc gat gct ggttat ctc caa 1200 Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala Gly TyrLeu Gln 385 390 395 400 gga att tac cgt acc ttg aaa gcc ata ttt ttc aatgaa atc aaa aga 1248 Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn GluIle Lys Arg 405 410 415 aat tcc aac ttg tat ttg tat agg tta tca gat gatacg tat agt gta 1296 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp ThrTyr Ser Val 420 425 430 tat aaa agt tat atc ttg ccc tat cga tgg ggt tccttg cca gga gtt 1344 Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser LeuPro Gly Val 435 440 445 agt cat ggt gat gat tta gga tat ctt ttt gca aactct ttg gat gtt 1392 Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn SerLeu Asp Val 450 455 460 cct att ttg gga aca acg cac att tct ata ccg caagat gct atg cag 1440 Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln AspAla Met Gln 465 470 475 480 act ctg gaa agg atg gtc agg atc tgg acc aatttt gta aag aat gga 1488 Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn PheVal Lys Asn Gly 485 490 495 aaa cct aca tca aac act gaa gat gca tca tgtgat aca aaa aga cat 1536 Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys AspThr Lys Arg His 500 505 510 tta aac gac att ttt tgg gaa cca tac aac gacgaa gaa cca aaa tat 1584 Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp GluGlu Pro Lys Tyr 515 520 525 ttg gac atg gga aaa gaa cat ttt gaa atg aaaaat att ttg gaa cta 1632 Leu Asp Met Gly Lys Glu His Phe Glu Met Lys AsnIle Leu Glu Leu 530 535 540 aaa cgc atg atg ctt tgg gat gaa gtt tat agaaat gcg aat ttg cgg 1680 Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg AsnAla Asn Leu Arg 545 550 555 560 ttt aga gtc tgt aat gaa gaa agt att aga1710 Phe Arg Val Cys Asn Glu Glu Ser Ile Arg 565 570 34 1785 DNACtenocephalides felis exon (1)..(1785) 34 atg tta cct cac agt gca ttagtt tta ttt tta ttt ttt tta ttt ttc 48 Met Leu Pro His Ser Ala Leu ValLeu Phe Leu Phe Phe Leu Phe Phe 1 5 10 15 tta ttt aca cct gtc ttg tgcata cta tgg gat aac cta gat cag cat 96 Leu Phe Thr Pro Val Leu Cys IleLeu Trp Asp Asn Leu Asp Gln His 20 25 30 ttg tgc aga gtt caa ttt aac gggatc acg gaa gga aaa ccg ttc cga 144 Leu Cys Arg Val Gln Phe Asn Gly IleThr Glu Gly Lys Pro Phe Arg 35 40 45 tat aaa gat cat aaa aat gat gta tattgt tcc tat ttg gga att cct 192 Tyr Lys Asp His Lys Asn Asp Val Tyr CysSer Tyr Leu Gly Ile Pro 50 55 60 tat gca gaa ccg cct att gga cca ttg cgattt cag tct cca aaa cca 240 Tyr Ala Glu Pro Pro Ile Gly Pro Leu Arg PheGln Ser Pro Lys Pro 65 70 75 80 ata tca aat cca aaa aca gga ttc gtt caggct cgg tct tta gga gac 288 Ile Ser Asn Pro Lys Thr Gly Phe Val Gln AlaArg Ser Leu Gly Asp 85 90 95 aaa tgt ttc cag gaa agt cta ata tat tct tatgca gga agc gaa gat 336 Lys Cys Phe Gln Glu Ser Leu Ile Tyr Ser Tyr AlaGly Ser Glu Asp 100 105 110 tgc tta tat ctg aat ata ttc acg cca gag actgtt aat tct gcg aac 384 Cys Leu Tyr Leu Asn Ile Phe Thr Pro Glu Thr ValAsn Ser Ala Asn 115 120 125 aat aca aaa tat cct gta atg ttc tgg atc catgga ggc gca ttc aac 432 Asn Thr Lys Tyr Pro Val Met Phe Trp Ile His GlyGly Ala Phe Asn 130 135 140 caa gga tca gga tct tat aat ttt ttt gga cctgat tat ttg atc agg 480 Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro AspTyr Leu Ile Arg 145 150 155 160 gaa gga att att ttg gtc act atc aac tataga tta gga gtt ttc ggt 528 Glu Gly Ile Ile Leu Val Thr Ile Asn Tyr ArgLeu Gly Val Phe Gly 165 170 175 ttt cta tca gcg ccg gaa tgg gat atc catgga aat atg ggt cta aaa 576 Phe Leu Ser Ala Pro Glu Trp Asp Ile His GlyAsn Met Gly Leu Lys 180 185 190 gac cag aga ttg gca cta aaa tgg gtt tatgac aac atc gaa aaa ttt 624 Asp Gln Arg Leu Ala Leu Lys Trp Val Tyr AspAsn Ile Glu Lys Phe 195 200 205 ggt gga gac aga gat aaa atc act ata gctgga gaa tct gct gga gca 672 Gly Gly Asp Arg Asp Lys Ile Thr Ile Ala GlyGlu Ser Ala Gly Ala 210 215 220 gca agt gtt cat ttt ctg atg atg gac aattct act aga aaa tac tac 720 Ala Ser Val His Phe Leu Met Met Asp Asn SerThr Arg Lys Tyr Tyr 225 230 235 240 caa agg gca att ttg cag agt ggg acatta ctc aat ccg act gct aat 768 Gln Arg Ala Ile Leu Gln Ser Gly Thr LeuLeu Asn Pro Thr Ala Asn 245 250 255 caa att caa cct ctg cat aga ttt gaaaaa cta aaa caa gtg ctg aac 816 Gln Ile Gln Pro Leu His Arg Phe Glu LysLeu Lys Gln Val Leu Asn 260 265 270 atc acg caa aaa caa gaa ctc cta aatctg gac aaa aat caa att ttg 864 Ile Thr Gln Lys Gln Glu Leu Leu Asn LeuAsp Lys Asn Gln Ile Leu 275 280 285 cga gca gcc tta aac aga gtc cca gataac aac gac cac gaa agg gac 912 Arg Ala Ala Leu Asn Arg Val Pro Asp AsnAsn Asp His Glu Arg Asp 290 295 300 aca gta cca gta ttt aat cca gtc ctagaa tca cca gaa tct cca gac 960 Thr Val Pro Val Phe Asn Pro Val Leu GluSer Pro Glu Ser Pro Asp 305 310 315 320 cca ata aca ttt cca tct gct ttagaa aga atg aga aat ggt gaa ttt 1008 Pro Ile Thr Phe Pro Ser Ala Leu GluArg Met Arg Asn Gly Glu Phe 325 330 335 cct gac gtt gat gtc atc att ggattc aat agt gct gaa ggt tta aga 1056 Pro Asp Val Asp Val Ile Ile Gly PheAsn Ser Ala Glu Gly Leu Arg 340 345 350 tct atg cca aga gta acc aga ggaaac atg gaa gtt tac aag act ttg 1104 Ser Met Pro Arg Val Thr Arg Gly AsnMet Glu Val Tyr Lys Thr Leu 355 360 365 aca aat ata gag aga gct ata cctaga gat gct aat att tgg aaa aat 1152 Thr Asn Ile Glu Arg Ala Ile Pro ArgAsp Ala Asn Ile Trp Lys Asn 370 375 380 cct aat ggc att gag gag aaa aaactt ata aaa atg ctt aca gag ttt 1200 Pro Asn Gly Ile Glu Glu Lys Lys LeuIle Lys Met Leu Thr Glu Phe 385 390 395 400 tat gac caa gtt aaa gaa caaaac gat gac atc gaa gcc tat gtc caa 1248 Tyr Asp Gln Val Lys Glu Gln AsnAsp Asp Ile Glu Ala Tyr Val Gln 405 410 415 cta aaa ggc gat gct ggt tatctc caa gga att tac cgt acc ttg aaa 1296 Leu Lys Gly Asp Ala Gly Tyr LeuGln Gly Ile Tyr Arg Thr Leu Lys 420 425 430 gcc ata ttt ttc aat gaa atcaaa aga aat tcc aac ttg tat ttg tat 1344 Ala Ile Phe Phe Asn Glu Ile LysArg Asn Ser Asn Leu Tyr Leu Tyr 435 440 445 agg tta tca gat gat acg tatagt gta tat aaa agt tat atc ttg ccc 1392 Arg Leu Ser Asp Asp Thr Tyr SerVal Tyr Lys Ser Tyr Ile Leu Pro 450 455 460 tat cga tgg ggt tcc ttg ccagga gtt agt cat ggt gat gat tta gga 1440 Tyr Arg Trp Gly Ser Leu Pro GlyVal Ser His Gly Asp Asp Leu Gly 465 470 475 480 tat ctt ttt gca aac tctttg gat gtt cct att ttg gga aca acg cac 1488 Tyr Leu Phe Ala Asn Ser LeuAsp Val Pro Ile Leu Gly Thr Thr His 485 490 495 att tct ata ccg caa gatgct atg cag act ctg gaa agg atg gtc agg 1536 Ile Ser Ile Pro Gln Asp AlaMet Gln Thr Leu Glu Arg Met Val Arg 500 505 510 atc tgg acc aat ttt gtaaag aat gga aaa cct aca tca aac act gaa 1584 Ile Trp Thr Asn Phe Val LysAsn Gly Lys Pro Thr Ser Asn Thr Glu 515 520 525 gat gca tca tgt gat acaaaa aga cat tta aac gac att ttt tgg gaa 1632 Asp Ala Ser Cys Asp Thr LysArg His Leu Asn Asp Ile Phe Trp Glu 530 535 540 cca tac aac gac gaa gaacca aaa tat ttg gac atg gga aaa gaa cat 1680 Pro Tyr Asn Asp Glu Glu ProLys Tyr Leu Asp Met Gly Lys Glu His 545 550 555 560 ttt gaa atg aaa aatatt ttg gaa cta aaa cgc atg atg ctt tgg gat 1728 Phe Glu Met Lys Asn IleLeu Glu Leu Lys Arg Met Met Leu Trp Asp 565 570 575 gaa gtt tat aga aatgcg aat ttg cgg ttt aga gtc tgt aat gaa gaa 1776 Glu Val Tyr Arg Asn AlaAsn Leu Arg Phe Arg Val Cys Asn Glu Glu 580 585 590 agt att aga 1785 SerIle Arg 595 35 1785 DNA Ctenocephalides felis 35 tctaatactt tcttcattacagactctaaa ccgcaaattc gcatttctat aaacttcatc 60 ccaaagcatc atgcgttttagttccaaaat atttttcatt tcaaaatgtt cttttcccat 120 gtccaaatat tttggttcttcgtcgttgta tggttcccaa aaaatgtcgt ttaaatgtct 180 ttttgtatca catgatgcatcttcagtgtt tgatgtaggt tttccattct ttacaaaatt 240 ggtccagatc ctgaccatcctttccagagt ctgcatagca tcttgcggta tagaaatgtg 300 cgttgttccc aaaataggaacatccaaaga gtttgcaaaa agatatccta aatcatcacc 360 atgactaact cctggcaaggaaccccatcg atagggcaag atataacttt tatatacact 420 atacgtatca tctgataacctatacaaata caagttggaa tttcttttga tttcattgaa 480 aaatatggct ttcaaggtacggtaaattcc ttggagataa ccagcatcgc cttttagttg 540 gacataggct tcgatgtcatcgttttgttc tttaacttgg tcataaaact ctgtaagcat 600 ttttataagt tttttctcctcaatgccatt aggatttttc caaatattag catctctagg 660 tatagctctc tctatatttgtcaaagtctt gtaaacttcc atgtttcctc tggttactct 720 tggcatagat cttaaaccttcagcactatt gaatccaatg atgacatcaa cgtcaggaaa 780 ttcaccattt ctcattctttctaaagcaga tggaaatgtt attgggtctg gagattctgg 840 tgattctagg actggattaaatactggtac tgtgtccctt tcgtggtcgt tgttatctgg 900 gactctgttt aaggctgctcgcaaaatttg atttttgtcc agatttagga gttcttgttt 960 ttgcgtgatg ttcagcacttgttttagttt ttcaaatcta tgcagaggtt gaatttgatt 1020 agcagtcgga ttgagtaatgtcccactctg caaaattgcc ctttggtagt attttctagt 1080 agaattgtcc atcatcagaaaatgaacact tgctgctcca gcagattctc cagctatagt 1140 gattttatct ctgtctccaccaaatttttc gatgttgtca taaacccatt ttagtgccaa 1200 tctctggtct tttagacccatatttccatg gatatcccat tccggcgctg atagaaaacc 1260 gaaaactcct aatctatagttgatagtgac caaaataatt ccttccctga tcaaataatc 1320 aggtccaaaa aaattataagatcctgatcc ttggttgaat gcgcctccat ggatccagaa 1380 cattacagga tattttgtattgttcgcaga attaacagtc tctggcgtga atatattcag 1440 atataagcaa tcttcgcttcctgcataaga atatattaga ctttcctgga aacatttgtc 1500 tcctaaagac cgagcctgaacgaatcctgt ttttggattt gatattggtt ttggagactg 1560 aaatcgcaat ggtccaataggcggttctgc ataaggaatt cccaaatagg aacaatatac 1620 atcattttta tgatctttatatcggaacgg ttttccttcc gtgatcccgt taaattgaac 1680 tctgcacaaa tgctgatctaggttatccca tagtatgcac aagacaggtg taaataagaa 1740 aaataaaaaa aataaaaataaaactaatgc actgtgaggt aacat 1785 36 2007 DNA Ctenocephalides felis CDS(11)..(1594) 36 agttccaacg atg gct gat cta caa gtg act ttg ctt caa ggtact tta 49 Met Ala Asp Leu Gln Val Thr Leu Leu Gln Gly Thr Leu 1 5 10aaa gga aaa gag caa att agt gaa aaa gga aat gtg ttc cat agt tat 97 LysGly Lys Glu Gln Ile Ser Glu Lys Gly Asn Val Phe His Ser Tyr 15 20 25 tctgga att cca tat gcc aaa cct cct gta ggt gat cta aga ttt aag 145 Ser GlyIle Pro Tyr Ala Lys Pro Pro Val Gly Asp Leu Arg Phe Lys 30 35 40 45 ccacct caa cct gca gaa cct tgg tca ggt gtt ctt gat gct agt aaa 193 Pro ProGln Pro Ala Glu Pro Trp Ser Gly Val Leu Asp Ala Ser Lys 50 55 60 gaa gggaat agt tgt aga tca gta cat ttt att aaa aaa att aaa gta 241 Glu Gly AsnSer Cys Arg Ser Val His Phe Ile Lys Lys Ile Lys Val 65 70 75 ggg gct gaagat tgt tta tac ctc aat gtc tat gta cca aaa aca tca 289 Gly Ala Glu AspCys Leu Tyr Leu Asn Val Tyr Val Pro Lys Thr Ser 80 85 90 gag aaa tca cttctt cca gta atg gta tgg ata cat gga gga ggc ttc 337 Glu Lys Ser Leu LeuPro Val Met Val Trp Ile His Gly Gly Gly Phe 95 100 105 ttc atg gga tctgga aat agt gat atg tat ggt cct gaa tat ttg atg 385 Phe Met Gly Ser GlyAsn Ser Asp Met Tyr Gly Pro Glu Tyr Leu Met 110 115 120 125 gat tat ggaatt gtt ctg gtt act ttc aat tat cga tta ggt gtt ttg 433 Asp Tyr Gly IleVal Leu Val Thr Phe Asn Tyr Arg Leu Gly Val Leu 130 135 140 gga ttt ttgaac ctg gga ata gaa gaa gcg cct ggc aat gtt ggt ttg 481 Gly Phe Leu AsnLeu Gly Ile Glu Glu Ala Pro Gly Asn Val Gly Leu 145 150 155 atg gac caggtt gaa gct cta aaa tgg gta aaa aac aat att gca tcc 529 Met Asp Gln ValGlu Ala Leu Lys Trp Val Lys Asn Asn Ile Ala Ser 160 165 170 ttt ggt ggtgac ccc aac aat gtg act att ttt gga gaa tca gca ggt 577 Phe Gly Gly AspPro Asn Asn Val Thr Ile Phe Gly Glu Ser Ala Gly 175 180 185 ggt gca agtgtt cat tat ttg atg tta tca gat ctt tcc aaa gga ctt 625 Gly Ala Ser ValHis Tyr Leu Met Leu Ser Asp Leu Ser Lys Gly Leu 190 195 200 205 ttt cataaa gcg atc tca caa agt gga agt gct ttt aat cct tgg gca 673 Phe His LysAla Ile Ser Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala 210 215 220 ctt caacat gat aat aat aaa gaa aat gca ttc cgc ctc tgc aaa ctt 721 Leu Gln HisAsp Asn Asn Lys Glu Asn Ala Phe Arg Leu Cys Lys Leu 225 230 235 ctg ggtcat cct gtc gat aac gag aca gaa gct cta aaa atc ctt cgt 769 Leu Gly HisPro Val Asp Asn Glu Thr Glu Ala Leu Lys Ile Leu Arg 240 245 250 caa gccccc ata gat gat ctt ata gac aac aga ata aaa cca aaa gac 817 Gln Ala ProIle Asp Asp Leu Ile Asp Asn Arg Ile Lys Pro Lys Asp 255 260 265 aaa ggccaa ctt att ata gac tat cct ttt cta cca aca ata gaa aaa 865 Lys Gly GlnLeu Ile Ile Asp Tyr Pro Phe Leu Pro Thr Ile Glu Lys 270 275 280 285 cgttat caa aat ttt gaa cca ttc ttg gac cag tct cca tta tca aaa 913 Arg TyrGln Asn Phe Glu Pro Phe Leu Asp Gln Ser Pro Leu Ser Lys 290 295 300 atgcaa tca ggc aat ttc aca aaa gtc cca ttt ata tgt gga tac aac 961 Met GlnSer Gly Asn Phe Thr Lys Val Pro Phe Ile Cys Gly Tyr Asn 305 310 315 agtgct gaa gga att tta ggt tta atg gac ttc aag gat gac cca aat 1009 Ser AlaGlu Gly Ile Leu Gly Leu Met Asp Phe Lys Asp Asp Pro Asn 320 325 330 atattt gag aag ttt gaa gct gat ttt gaa aga ttt gta cca gta gat 1057 Ile PheGlu Lys Phe Glu Ala Asp Phe Glu Arg Phe Val Pro Val Asp 335 340 345 ttgaat cta act tta agg tct aag gaa tct aaa aaa ttg gct gaa gaa 1105 Leu AsnLeu Thr Leu Arg Ser Lys Glu Ser Lys Lys Leu Ala Glu Glu 350 355 360 365atg aga aag ttt tat tac caa gac gaa cct gtt tct tca gac aac aaa 1153 MetArg Lys Phe Tyr Tyr Gln Asp Glu Pro Val Ser Ser Asp Asn Lys 370 375 380gaa aaa ttt gtc agt gtt att agt gat act tgg ttt ttg aga ggg att 1201 GluLys Phe Val Ser Val Ile Ser Asp Thr Trp Phe Leu Arg Gly Ile 385 390 395aaa aat act gca aga tat ata att gaa cat tcc tca gaa ccg tta tat 1249 LysAsn Thr Ala Arg Tyr Ile Ile Glu His Ser Ser Glu Pro Leu Tyr 400 405 410tta tat gtt tat agt ttt gat gat ttt ggt ttt ttg aag aaa ctt gta 1297 LeuTyr Val Tyr Ser Phe Asp Asp Phe Gly Phe Leu Lys Lys Leu Val 415 420 425tta gat cct aat att gaa gga gca gct cat gga gat gag ctg gga tat 1345 LeuAsp Pro Asn Ile Glu Gly Ala Ala His Gly Asp Glu Leu Gly Tyr 430 435 440445 ctt ttc aag atg agt ttt aca gaa ttt cca aaa gat tta cca agt gca 1393Leu Phe Lys Met Ser Phe Thr Glu Phe Pro Lys Asp Leu Pro Ser Ala 450 455460 gtg gtg aat agg gaa cga ttg ttg caa ctt tgg aca aat ttt gca aaa 1441Val Val Asn Arg Glu Arg Leu Leu Gln Leu Trp Thr Asn Phe Ala Lys 465 470475 aca gga aat ccc act cct gaa atc aat gat gtt ata aca aca aaa tgg 1489Thr Gly Asn Pro Thr Pro Glu Ile Asn Asp Val Ile Thr Thr Lys Trp 480 485490 gat aaa gct act gag gaa aaa tca gat cat atg gat atc gat aat act 1537Asp Lys Ala Thr Glu Glu Lys Ser Asp His Met Asp Ile Asp Asn Thr 495 500505 ttg aga atg att cca gat cct gat gca aaa cga ctt aga ttt tgg aat 1585Leu Arg Met Ile Pro Asp Pro Asp Ala Lys Arg Leu Arg Phe Trp Asn 510 515520 525 aaa ttt tta tgataaatat accaattatc gattttatta tagagtttct 1634 LysPhe Leu gtattagtat aattatcacg tttagatgta cgagattcaa ttggctctaattgaagtata 1694 tttcgatttc aaatttactc tgattattgg aaaaaaagct tttacagttgtaataatcaa 1754 gaagtaggtg gtaaatttag aacaaattct gttttagtga tttgcgcattcaacagatgg 1814 tgtactgtgc ctaaatttgt cgctcttctt gaagaactga actaaaaatgtgattaatgg 1874 acgccacatt atttatattt gatattatta ccatctttgt atcatatttgcttttatttt 1934 ttcatttttt ttttatttca aatatattgt ttttttataa aaaaaaaaaaaaaaaaaaaa 1994 aaaaaaaaaa aaa 2007 37 528 PRT Ctenocephalides felis 37Met Ala Asp Leu Gln Val Thr Leu Leu Gln Gly Thr Leu Lys Gly Lys 1 5 1015 Glu Gln Ile Ser Glu Lys Gly Asn Val Phe His Ser Tyr Ser Gly Ile 20 2530 Pro Tyr Ala Lys Pro Pro Val Gly Asp Leu Arg Phe Lys Pro Pro Gln 35 4045 Pro Ala Glu Pro Trp Ser Gly Val Leu Asp Ala Ser Lys Glu Gly Asn 50 5560 Ser Cys Arg Ser Val His Phe Ile Lys Lys Ile Lys Val Gly Ala Glu 65 7075 80 Asp Cys Leu Tyr Leu Asn Val Tyr Val Pro Lys Thr Ser Glu Lys Ser 8590 95 Leu Leu Pro Val Met Val Trp Ile His Gly Gly Gly Phe Phe Met Gly100 105 110 Ser Gly Asn Ser Asp Met Tyr Gly Pro Glu Tyr Leu Met Asp TyrGly 115 120 125 Ile Val Leu Val Thr Phe Asn Tyr Arg Leu Gly Val Leu GlyPhe Leu 130 135 140 Asn Leu Gly Ile Glu Glu Ala Pro Gly Asn Val Gly LeuMet Asp Gln 145 150 155 160 Val Glu Ala Leu Lys Trp Val Lys Asn Asn IleAla Ser Phe Gly Gly 165 170 175 Asp Pro Asn Asn Val Thr Ile Phe Gly GluSer Ala Gly Gly Ala Ser 180 185 190 Val His Tyr Leu Met Leu Ser Asp LeuSer Lys Gly Leu Phe His Lys 195 200 205 Ala Ile Ser Gln Ser Gly Ser AlaPhe Asn Pro Trp Ala Leu Gln His 210 215 220 Asp Asn Asn Lys Glu Asn AlaPhe Arg Leu Cys Lys Leu Leu Gly His 225 230 235 240 Pro Val Asp Asn GluThr Glu Ala Leu Lys Ile Leu Arg Gln Ala Pro 245 250 255 Ile Asp Asp LeuIle Asp Asn Arg Ile Lys Pro Lys Asp Lys Gly Gln 260 265 270 Leu Ile IleAsp Tyr Pro Phe Leu Pro Thr Ile Glu Lys Arg Tyr Gln 275 280 285 Asn PheGlu Pro Phe Leu Asp Gln Ser Pro Leu Ser Lys Met Gln Ser 290 295 300 GlyAsn Phe Thr Lys Val Pro Phe Ile Cys Gly Tyr Asn Ser Ala Glu 305 310 315320 Gly Ile Leu Gly Leu Met Asp Phe Lys Asp Asp Pro Asn Ile Phe Glu 325330 335 Lys Phe Glu Ala Asp Phe Glu Arg Phe Val Pro Val Asp Leu Asn Leu340 345 350 Thr Leu Arg Ser Lys Glu Ser Lys Lys Leu Ala Glu Glu Met ArgLys 355 360 365 Phe Tyr Tyr Gln Asp Glu Pro Val Ser Ser Asp Asn Lys GluLys Phe 370 375 380 Val Ser Val Ile Ser Asp Thr Trp Phe Leu Arg Gly IleLys Asn Thr 385 390 395 400 Ala Arg Tyr Ile Ile Glu His Ser Ser Glu ProLeu Tyr Leu Tyr Val 405 410 415 Tyr Ser Phe Asp Asp Phe Gly Phe Leu LysLys Leu Val Leu Asp Pro 420 425 430 Asn Ile Glu Gly Ala Ala His Gly AspGlu Leu Gly Tyr Leu Phe Lys 435 440 445 Met Ser Phe Thr Glu Phe Pro LysAsp Leu Pro Ser Ala Val Val Asn 450 455 460 Arg Glu Arg Leu Leu Gln LeuTrp Thr Asn Phe Ala Lys Thr Gly Asn 465 470 475 480 Pro Thr Pro Glu IleAsn Asp Val Ile Thr Thr Lys Trp Asp Lys Ala 485 490 495 Thr Glu Glu LysSer Asp His Met Asp Ile Asp Asn Thr Leu Arg Met 500 505 510 Ile Pro AspPro Asp Ala Lys Arg Leu Arg Phe Trp Asn Lys Phe Leu 515 520 525 38 2007DNA Ctenocephalides felis 38 tttttttttt tttttttttt tttttttttt tttttataaaaaaacaatat atttgaaata 60 aaaaaaaaat gaaaaaataa aagcaaatat gatacaaagatggtaataat atcaaatata 120 aataatgtgg cgtccattaa tcacattttt agttcagttcttcaagaaga gcgacaaatt 180 taggcacagt acaccatctg ttgaatgcgc aaatcactaaaacagaattt gttctaaatt 240 taccacctac ttcttgatta ttacaactgt aaaagctttttttccaataa tcagagtaaa 300 tttgaaatcg aaatatactt caattagagc caattgaatctcgtacatct aaacgtgata 360 attatactaa tacagaaact ctataataaa atcgataattggtatattta tcataaaaat 420 ttattccaaa atctaagtcg ttttgcatca ggatctggaatcattctcaa agtattatcg 480 atatccatat gatctgattt ttcctcagta gctttatcccattttgttgt tataacatca 540 ttgatttcag gagtgggatt tcctgttttt gcaaaatttgtccaaagttg caacaatcgt 600 tccctattca ccactgcact tggtaaatct tttggaaattctgtaaaact catcttgaaa 660 agatatccca gctcatctcc atgagctgct ccttcaatattaggatctaa tacaagtttc 720 ttcaaaaaac caaaatcatc aaaactataa acatataaatataacggttc tgaggaatgt 780 tcaattatat atcttgcagt atttttaatc cctctcaaaaaccaagtatc actaataaca 840 ctgacaaatt tttctttgtt gtctgaagaa acaggttcgtcttggtaata aaactttctc 900 atttcttcag ccaatttttt agattcctta gaccttaaagttagattcaa atctactggt 960 acaaatcttt caaaatcagc ttcaaacttc tcaaatatatttgggtcatc cttgaagtcc 1020 attaaaccta aaattccttc agcactgttg tatccacatataaatgggac ttttgtgaaa 1080 ttgcctgatt gcatttttga taatggagac tggtccaagaatggttcaaa attttgataa 1140 cgtttttcta ttgttggtag aaaaggatag tctataataagttggccttt gtcttttggt 1200 tttattctgt tgtctataag atcatctatg ggggcttgacgaaggatttt tagagcttct 1260 gtctcgttat cgacaggatg acccagaagt ttgcagaggcggaatgcatt ttctttatta 1320 ttatcatgtt gaagtgccca aggattaaaa gcacttccactttgtgagat cgctttatga 1380 aaaagtcctt tggaaagatc tgataacatc aaataatgaacacttgcacc acctgctgat 1440 tctccaaaaa tagtcacatt gttggggtca ccaccaaaggatgcaatatt gttttttacc 1500 cattttagag cttcaacctg gtccatcaaa ccaacattgccaggcgcttc ttctattccc 1560 aggttcaaaa atcccaaaac acctaatcga taattgaaagtaaccagaac aattccataa 1620 tccatcaaat attcaggacc atacatatca ctatttccagatcccatgaa gaagcctcct 1680 ccatgtatcc ataccattac tggaagaagt gatttctctgatgtttttgg tacatagaca 1740 ttgaggtata aacaatcttc agcccctact ttaatttttttaataaaatg tactgatcta 1800 caactattcc cttctttact agcatcaaga acacctgaccaaggttctgc aggttgaggt 1860 ggcttaaatc ttagatcacc tacaggaggt ttggcatatggaattccaga ataactatgg 1920 aacacatttc ctttttcact aatttgctct tttccttttaaagtaccttg aagcaaagtc 1980 acttgtagat cagccatcgt tggaact 2007 39 12 PRTPeptide 39 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu 1 5 10 40 22PRT Peptide MISC_FEATURE (21)..(21) Xaa = unknown 40 Asp Pro Pro Thr ValThr Leu Pro Gln Gly Glu Leu Val Gly Lys Ala 1 5 10 15 Thr Asn Glu AsnXaa Lys 20 41 12 PRT Peptide 41 Asp Pro Pro Thr Val Thr Leu Pro Gln GlyGlu Leu 1 5 10 42 21 PRT Peptide 42 Asp Pro Pro Thr Val Thr Leu Pro GlnGly Glu Leu Val Gly Lys Ala 1 5 10 15 Leu Ser Asn Glu Asn 20 43 8 PRTPeptide 43 Asp Pro Pro Thr Val Thr Leu Pro 1 5 44 23 PRT Peptide 44 AspPro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val Gly Lys Ala 1 5 10 15Leu Thr Asn Glu Asn Gly Lys 20 45 20 DNA Artificial sequence SyntheticPrimer 45 aattaaccct cactaaaggg 20 46 17 DNA Artificial sequenceSynthetic Primer 46 ardccdccdc crtrdat 17 47 38 DNA Artificial sequenceSynthetic Primer 47 tgtgctcgag atgggataac ctagatcagc atttgtgc 38 48 35DNA Artificial sequence Synthetic Primer 48 ttaaggtacc tcatctaatacttccttcat tacag 35 49 36 DNA Artificial sequence Synthetic Primer 49aaaactgcag tataaatatg ttacctcaca gtagtg 36 50 34 DNA Artificial sequenceSynthetic Primer 50 tgctctagat tatctaatac ttccttcatt acag 34 51 1584 DNACtenocephalides felis exon (1)..(1584) 51 atg gct gat cta caa gtg actttg ctt caa ggt act tta aaa gga aaa 48 Met Ala Asp Leu Gln Val Thr LeuLeu Gln Gly Thr Leu Lys Gly Lys 1 5 10 15 gag caa att agt gaa aaa ggaaat gtg ttc cat agt tat tct gga att 96 Glu Gln Ile Ser Glu Lys Gly AsnVal Phe His Ser Tyr Ser Gly Ile 20 25 30 cca tat gcc aaa cct cct gta ggtgat cta aga ttt aag cca cct caa 144 Pro Tyr Ala Lys Pro Pro Val Gly AspLeu Arg Phe Lys Pro Pro Gln 35 40 45 cct gca gaa cct tgg tca ggt gtt cttgat gct agt aaa gaa ggg aat 192 Pro Ala Glu Pro Trp Ser Gly Val Leu AspAla Ser Lys Glu Gly Asn 50 55 60 agt tgt aga tca gta cat ttt att aaa aaaatt aaa gta ggg gct gaa 240 Ser Cys Arg Ser Val His Phe Ile Lys Lys IleLys Val Gly Ala Glu 65 70 75 80 gat tgt tta tac ctc aat gtc tat gta ccaaaa aca tca gag aaa tca 288 Asp Cys Leu Tyr Leu Asn Val Tyr Val Pro LysThr Ser Glu Lys Ser 85 90 95 ctt ctt cca gta atg gta tgg ata cat gga ggaggc ttc ttc atg gga 336 Leu Leu Pro Val Met Val Trp Ile His Gly Gly GlyPhe Phe Met Gly 100 105 110 tct gga aat agt gat atg tat ggt cct gaa tatttg atg gat tat gga 384 Ser Gly Asn Ser Asp Met Tyr Gly Pro Glu Tyr LeuMet Asp Tyr Gly 115 120 125 att gtt ctg gtt act ttc aat tat cga tta ggtgtt ttg gga ttt ttg 432 Ile Val Leu Val Thr Phe Asn Tyr Arg Leu Gly ValLeu Gly Phe Leu 130 135 140 aac ctg gga ata gaa gaa gcg cct ggc aat gttggt ttg atg gac cag 480 Asn Leu Gly Ile Glu Glu Ala Pro Gly Asn Val GlyLeu Met Asp Gln 145 150 155 160 gtt gaa gct cta aaa tgg gta aaa aac aatatt gca tcc ttt ggt ggt 528 Val Glu Ala Leu Lys Trp Val Lys Asn Asn IleAla Ser Phe Gly Gly 165 170 175 gac ccc aac aat gtg act att ttt gga gaatca gca ggt ggt gca agt 576 Asp Pro Asn Asn Val Thr Ile Phe Gly Glu SerAla Gly Gly Ala Ser 180 185 190 gtt cat tat ttg atg tta tca gat ctt tccaaa gga ctt ttt cat aaa 624 Val His Tyr Leu Met Leu Ser Asp Leu Ser LysGly Leu Phe His Lys 195 200 205 gcg atc tca caa agt gga agt gct ttt aatcct tgg gca ctt caa cat 672 Ala Ile Ser Gln Ser Gly Ser Ala Phe Asn ProTrp Ala Leu Gln His 210 215 220 gat aat aat aaa gaa aat gca ttc cgc ctctgc aaa ctt ctg ggt cat 720 Asp Asn Asn Lys Glu Asn Ala Phe Arg Leu CysLys Leu Leu Gly His 225 230 235 240 cct gtc gat aac gag aca gaa gct ctaaaa atc ctt cgt caa gcc ccc 768 Pro Val Asp Asn Glu Thr Glu Ala Leu LysIle Leu Arg Gln Ala Pro 245 250 255 ata gat gat ctt ata gac aac aga ataaaa cca aaa gac aaa ggc caa 816 Ile Asp Asp Leu Ile Asp Asn Arg Ile LysPro Lys Asp Lys Gly Gln 260 265 270 ctt att ata gac tat cct ttt cta ccaaca ata gaa aaa cgt tat caa 864 Leu Ile Ile Asp Tyr Pro Phe Leu Pro ThrIle Glu Lys Arg Tyr Gln 275 280 285 aat ttt gaa cca ttc ttg gac cag tctcca tta tca aaa atg caa tca 912 Asn Phe Glu Pro Phe Leu Asp Gln Ser ProLeu Ser Lys Met Gln Ser 290 295 300 ggc aat ttc aca aaa gtc cca ttt atatgt gga tac aac agt gct gaa 960 Gly Asn Phe Thr Lys Val Pro Phe Ile CysGly Tyr Asn Ser Ala Glu 305 310 315 320 gga att tta ggt tta atg gac ttcaag gat gac cca aat ata ttt gag 1008 Gly Ile Leu Gly Leu Met Asp Phe LysAsp Asp Pro Asn Ile Phe Glu 325 330 335 aag ttt gaa gct gat ttt gaa agattt gta cca gta gat ttg aat cta 1056 Lys Phe Glu Ala Asp Phe Glu Arg PheVal Pro Val Asp Leu Asn Leu 340 345 350 act tta agg tct aag gaa tct aaaaaa ttg gct gaa gaa atg aga aag 1104 Thr Leu Arg Ser Lys Glu Ser Lys LysLeu Ala Glu Glu Met Arg Lys 355 360 365 ttt tat tac caa gac gaa cct gtttct tca gac aac aaa gaa aaa ttt 1152 Phe Tyr Tyr Gln Asp Glu Pro Val SerSer Asp Asn Lys Glu Lys Phe 370 375 380 gtc agt gtt att agt gat act tggttt ttg aga ggg att aaa aat act 1200 Val Ser Val Ile Ser Asp Thr Trp PheLeu Arg Gly Ile Lys Asn Thr 385 390 395 400 gca aga tat ata att gaa cattcc tca gaa ccg tta tat tta tat gtt 1248 Ala Arg Tyr Ile Ile Glu His SerSer Glu Pro Leu Tyr Leu Tyr Val 405 410 415 tat agt ttt gat gat ttt ggtttt ttg aag aaa ctt gta tta gat cct 1296 Tyr Ser Phe Asp Asp Phe Gly PheLeu Lys Lys Leu Val Leu Asp Pro 420 425 430 aat att gaa gga gca gct catgga gat gag ctg gga tat ctt ttc aag 1344 Asn Ile Glu Gly Ala Ala His GlyAsp Glu Leu Gly Tyr Leu Phe Lys 435 440 445 atg agt ttt aca gaa ttt ccaaaa gat tta cca agt gca gtg gtg aat 1392 Met Ser Phe Thr Glu Phe Pro LysAsp Leu Pro Ser Ala Val Val Asn 450 455 460 52 1584 DNA Ctenocephalidesfelis 52 taaaaattta ttccaaaatc taagtcgttt tgcatcagga tctggaatcattctcaaagt 60 attatcgata tccatatgat ctgatttttc ctcagtagct ttatcccattttgttgttat 120 aacatcattg atttcaggag tgggatttcc tgtttttgca aaatttgtccaaagttgcaa 180 caatcgttcc ctattcacca ctgcacttgg taaatctttt ggaaattctgtaaaactcat 240 cttgaaaaga tatcccagct catctccatg agctgctcct tcaatattaggatctaatac 300 aagtttcttc aaaaaaccaa aatcatcaaa actataaaca tataaatataacggttctga 360 ggaatgttca attatatatc ttgcagtatt tttaatccct ctcaaaaaccaagtatcact 420 aataacactg acaaattttt ctttgttgtc tgaagaaaca ggttcgtcttggtaataaaa 480 ctttctcatt tcttcagcca attttttaga ttccttagac cttaaagttagattcaaatc 540 tactggtaca aatctttcaa aatcagcttc aaacttctca aatatatttgggtcatcctt 600 gaagtccatt aaacctaaaa ttccttcagc actgttgtat ccacatataaatgggacttt 660 tgtgaaattg cctgattgca tttttgataa tggagactgg tccaagaatggttcaaaatt 720 ttgataacgt ttttctattg ttggtagaaa aggatagtct ataataagttggcctttgtc 780 ttttggtttt attctgttgt ctataagatc atctatgggg gcttgacgaaggatttttag 840 agcttctgtc tcgttatcga caggatgacc cagaagtttg cagaggcggaatgcattttc 900 tttattatta tcatgttgaa gtgcccaagg attaaaagca cttccactttgtgagatcgc 960 tttatgaaaa agtcctttgg aaagatctga taacatcaaa taatgaacacttgcaccacc 1020 tgctgattct ccaaaaatag tcacattgtt ggggtcacca ccaaaggatgcaatattgtt 1080 ttttacccat tttagagctt caacctggtc catcaaacca acattgccaggcgcttcttc 1140 tattcccagg ttcaaaaatc ccaaaacacc taatcgataa ttgaaagtaaccagaacaat 1200 tccataatcc atcaaatatt caggaccata catatcacta tttccagatcccatgaagaa 1260 gcctcctcca tgtatccata ccattactgg aagaagtgat ttctctgatgtttttggtac 1320 atagacattg aggtataaac aatcttcagc ccctacttta atttttttaataaaatgtac 1380 tgatctacaa ctattccctt ctttactagc atcaagaaca cctgaccaaggttctgcagg 1440 ttgaggtggc ttaaatctta gatcacctac aggaggtttg gcatatggaattccagaata 1500 actatggaac acatttcctt tttcactaat ttgctctttt ccttttaaagtaccttgaag 1560 caaagtcact tgtagatcag ccat 1584 53 530 PRTCtenocephalides felis 53 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu LeuVal Gly Lys Ala 1 5 10 15 Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe SerTyr Thr Gly Val Pro 20 25 30 Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg PheLys Pro Pro Gln Lys 35 40 45 Ala Glu Pro Trp Asn Gly Val Phe Asn Ala ThrSer His Gly Asn Val 50 55 60 Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys IleGlu Gly Asp Glu Asp 65 70 75 80 Cys Leu Leu Val Asn Val Tyr Ala Pro LysThr Thr Ser Asp Lys Lys 85 90 95 Leu Pro Val Phe Phe Trp Val His Gly GlyGly Phe Val Thr Gly Ser 100 105 110 Gly Asn Leu Glu Phe Gln Ser Pro AspTyr Leu Val Asn Tyr Asp Val 115 120 125 Ile Phe Val Thr Phe Asn Tyr ArgLeu Gly Pro Leu Gly Phe Leu Asn 130 135 140 Leu Glu Leu Glu Gly Ala ProGly Asn Val Gly Leu Leu Asp Gln Val 145 150 155 160 Ala Ala Leu Lys TrpThr Lys Glu Asn Ile Glu Lys Phe Gly Gly Asp 165 170 175 Pro Glu Asn IleThr Ile Gly Gly Val Ser Ala Gly Gly Ala Ser Val 180 185 190 His Tyr LeuLeu Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg Ala 195 200 205 Ile AlaGln Ser Gly Ser Ala Leu Asn Pro Trp Ala Phe Gln Arg His 210 215 220 ProVal Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu Gly His Pro Thr 225 230 235240 Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu Gln Lys Ala Pro Val Asp 245250 255 Ser Leu Leu Lys Lys Met Pro Ala Glu Thr Glu Gly Glu Ile Ile Glu260 265 270 Glu Phe Val Phe Val Pro Ser Ile Glu Lys Val Phe Pro Ser HisGln 275 280 285 Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys Ser GlySer Phe 290 295 300 Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala GluGly Leu Leu 305 310 315 320 Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu MetLeu Asn Gln Ala Glu 325 330 335 Ala Asp Phe Glu Arg Leu Val Pro Ala GluPhe Glu Leu Ala His Gly 340 345 350 Ser Glu Glu Ser Lys Lys Leu Ala GluLys Ile Arg Lys Phe Tyr Phe 355 360 365 Asp Asp Lys Pro Val Pro Glu AsnGlu Gln Lys Phe Ile Asp Leu Ile 370 375 380 Gly Asp Ile Trp Phe Thr ArgGly Ile Asp Lys His Val Lys Leu Ser 385 390 395 400 Val Glu Lys Gln AspGlu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser 405 410 415 Glu Ser His ProAla Lys Gly Thr Phe Gly Asp His Asn Leu Thr Gly 420 425 430 Ala Cys HisGly Glu Glu Leu Val Asn Leu Phe Lys Val Glu Met Met 435 440 445 Lys LeuGlu Lys Asp Lys Pro Asn Val Leu Leu Thr Lys Asp Arg Val 450 455 460 LeuAla Met Trp Thr Asn Phe Ile Lys Asn Gly Asn Pro Thr Pro Glu 465 470 475480 Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro Ala Thr Lys Asp Lys 485490 495 Leu Asn Tyr Leu Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn Pro500 505 510 Glu Glu Thr Arg Val Lys Phe Trp Glu Asp Ala Thr Lys Thr LeuHis 515 520 525 Ser Gln 530 54 570 PRT Ctenocephalides felis 54 Trp AspAsn Leu Asp Gln His Leu Cys Arg Val Gln Phe Asn Gly Ile 1 5 10 15 ThrGlu Gly Lys Pro Phe Arg Tyr Lys Asp His Arg Asn Asp Val Tyr 20 25 30 CysSer Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu 35 40 45 ArgPhe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 50 55 60 GlnAla Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr 65 70 75 80Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro 85 90 95Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val Met Phe Trp 100 105110 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe 115120 125 Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val Thr Ile Asn130 135 140 Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp AspIle 145 150 155 160 His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala LeuLys Trp Val 165 170 175 Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg GluLys Ile Thr Ile 180 185 190 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val HisPhe Leu Met Met Asp 195 200 205 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg AlaIle Leu Gln Ser Gly Thr 210 215 220 Leu Leu Asn Pro Thr Ala Asn Gln IleGln Leu Leu His Arg Phe Glu 225 230 235 240 Lys Leu Lys Gln Val Leu AsnIle Thr Gln Lys Gln Glu Leu Leu Asn 245 250 255 Leu Asp Lys Asn Leu IleLeu Arg Ala Ala Leu Asn Arg Val Pro Asp 260 265 270 Ser Asn Asp His AspArg Asp Thr Val Pro Val Phe Asn Pro Val Leu 275 280 285 Glu Ser Pro GluSer Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290 295 300 Arg Met ArgAsn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 305 310 315 320 AsnSer Ala Glu Gly Leu Arg Ser Met Ala Arg Val Thr Arg Gly Asn 325 330 335Met Glu Val His Lys Thr Leu Thr Asn Ile Glu Arg Ala Ile Pro Arg 340 345350 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu Glu Lys Lys Leu 355360 365 Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys Glu Gln Asn Asp370 375 380 Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala Gly Tyr LeuGln 385 390 395 400 Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn GluPhe Arg Arg 405 410 415 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp AspThr Tyr Ser Val 420 425 430 Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp GlySer Leu Pro Gly Val 435 440 445 Ser His Gly Asp Asp Leu Gly Tyr Leu PheAla Asn Ser Leu Asp Val 450 455 460 Pro Ile Leu Gly Thr Thr His Ile SerIle Pro Gln Asp Ala Met Gln 465 470 475 480 Thr Leu Glu Arg Met Val ArgIle Trp Thr Asn Phe Val Lys Asn Gly 485 490 495 Lys Pro Thr Ser Asn ThrGlu Asp Ala Ser Cys Asp Thr Lys Arg His 500 505 510 Leu Asn Asp Ile PheTrp Glu Pro Tyr Asn Asp Glu Glu Pro Lys Tyr 515 520 525 Leu Asp Met GlyLys Glu Asn Phe Glu Met Lys Asn Ile Leu Glu Leu 530 535 540 Lys Arg MetMet Leu Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg 545 550 555 560 PheArg Val Cys Asn Glu Gly Ser Ile Arg 565 570 55 570 PRT Ctenocephalidesfelis 55 Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln Phe Asn Gly Ile1 5 10 15 Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Lys Asn Asp ValTyr 20 25 30 Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Ile Gly ProLeu 35 40 45 Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly PheVal 50 55 60 Gln Ala Arg Ser Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu IleTyr 65 70 75 80 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile PheThr Pro 85 90 95 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val MetPhe Trp 100 105 110 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser TyrAsn Phe Phe 115 120 125 Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile LeuVal Thr Ile Asn 130 135 140 Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser AlaPro Glu Trp Asp Ile 145 150 155 160 His Gly Asn Met Gly Leu Lys Asp GlnArg Leu Ala Leu Lys Trp Val 165 170 175 Tyr Asp Asn Ile Glu Lys Phe GlyGly Asp Arg Asp Lys Ile Thr Ile 180 185 190 Ala Gly Glu Ser Ala Gly AlaAla Ser Val His Phe Leu Met Met Asp 195 200 205 Asn Ser Thr Arg Lys TyrTyr Gln Arg Ala Ile Leu Gln Ser Gly Thr 210 215 220 Leu Leu Asn Pro ThrAla Asn Gln Ile Gln Pro Leu His Arg Phe Glu 225 230 235 240 Lys Leu LysGln Val Leu Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn 245 250 255 Leu AspLys Asn Gln Ile Leu Arg Ala Ala Leu Asn Arg Val Pro Asp 260 265 270 AsnAsn Asp His Glu Arg Asp Thr Val Pro Val Phe Asn Pro Val Leu 275 280 285Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290 295300 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 305310 315 320 Asn Ser Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr Arg GlyAsn 325 330 335 Met Glu Val Tyr Lys Thr Leu Thr Asn Ile Glu Arg Ala IlePro Arg 340 345 350 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu GluLys Lys Leu 355 360 365 Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val LysGlu Gln Asn Asp 370 375 380 Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly AspAla Gly Tyr Leu Gln 385 390 395 400 Gly Ile Tyr Arg Thr Leu Lys Ala IlePhe Phe Asn Glu Ile Lys Arg 405 410 415 Asn Ser Asn Leu Tyr Leu Tyr ArgLeu Ser Asp Asp Thr Tyr Ser Val 420 425 430 Tyr Lys Ser Tyr Ile Leu ProTyr Arg Trp Gly Ser Leu Pro Gly Val 435 440 445 Ser His Gly Asp Asp LeuGly Tyr Leu Phe Ala Asn Ser Leu Asp Val 450 455 460 Pro Ile Leu Gly ThrThr His Ile Ser Ile Pro Gln Asp Ala Met Gln 465 470 475 480 Thr Leu GluArg Met Val Arg Ile Trp Thr Asn Phe Val Lys Asn Gly 485 490 495 Lys ProThr Ser Asn Thr Glu Asp Ala Ser Cys Asp Thr Lys Arg His 500 505 510 LeuAsn Asp Ile Phe Trp Glu Pro Tyr Asn Asp Glu Glu Pro Lys Tyr 515 520 525Leu Asp Met Gly Lys Glu His Phe Glu Met Lys Asn Ile Leu Glu Leu 530 535540 Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg 545550 555 560 Phe Arg Val Cys Asn Glu Gly Ser Ile Arg 565 570 56 20 DNAArtificial sequence Synthetic Primer 56 gtgcgtacac gtttactacc 20 57 2144DNA Ctenocephalides felis CDS (30)..(1682) 57 gtacacatag tcaatagtctagatccaag atg tct cgt gtt att ttt tta agt 53 Met Ser Arg Val Ile Phe LeuSer 1 5 tgt att ttt ttg ttt agt ttt aat ttt ata aaa tgt gat tcc ccg act101 Cys Ile Phe Leu Phe Ser Phe Asn Phe Ile Lys Cys Asp Ser Pro Thr 1015 20 gta act ttg ccc caa ggc gaa ttg gtt gga aaa gct ttg acg aac gaa149 Val Thr Leu Pro Gln Gly Glu Leu Val Gly Lys Ala Leu Thr Asn Glu 2530 35 40 aat gga aaa gag tat ttt agc tac aca ggt gta cct tat gct aaa cct197 Asn Gly Lys Glu Tyr Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys Pro 4550 55 cct gtt gga gaa ctt aga ttt aag cct cca cag aaa gct gag cca tgg245 Pro Val Gly Glu Leu Arg Phe Lys Pro Pro Gln Lys Ala Glu Pro Trp 6065 70 caa ggt gtt ttc aac gcc aca tta tac gga aat gtg tgt aaa tct tta293 Gln Gly Val Phe Asn Ala Thr Leu Tyr Gly Asn Val Cys Lys Ser Leu 7580 85 aat ttc ttc ttg aag aaa att gaa gga gac gaa gac tgc ttg gta gta341 Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp Glu Asp Cys Leu Val Val 9095 100 aac gtg tac gca cca aaa aca act tct gat aaa aaa ctt cca gta ttt389 Asn Val Tyr Ala Pro Lys Thr Thr Ser Asp Lys Lys Leu Pro Val Phe 105110 115 120 ttc tgg gtt cat ggt ggt ggt ttt gtg act gga tcc gga aat ttagaa 437 Phe Trp Val His Gly Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu125 130 135 ttc caa agc cca gat tat tta gta rat ttt gat gtt att ttc gtaact 485 Phe Gln Ser Pro Asp Tyr Leu Val Xaa Phe Asp Val Ile Phe Val Thr140 145 150 ttc aat tac cga ttg gga cct ctc gga ttt ctg aat ttg gag ttggag 533 Phe Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu Asn Leu Glu Leu Glu155 160 165 ggt gct cca gga aat gta gga tta ttg gat cag gtg gca gct ctgaaa 581 Gly Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Val Ala Ala Leu Lys170 175 180 tgg acc aaa gaa aac att gag aaa ttt ggt gga gat cca gaa aatatt 629 Trp Thr Lys Glu Asn Ile Glu Lys Phe Gly Gly Asp Pro Glu Asn Ile185 190 195 200 aca att ggt ggt gtt tct gct ggt gga gca agt gtt cat tatctt ttg 677 Thr Ile Gly Gly Val Ser Ala Gly Gly Ala Ser Val His Tyr LeuLeu 205 210 215 tta tct cat aca acc act gga ctt tac aaa agg gca att gctcaa agt 725 Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg Ala Ile Ala GlnSer 220 225 230 gga agt gct ttt aat cca tgg gcc ttc caa aga cat cca gtaaag cgt 773 Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln Arg His Pro Val LysArg 235 240 245 agt ctt caa ctt gct gag ata ttg ggt cat ccc aca aac aatact caa 821 Ser Leu Gln Leu Ala Glu Ile Leu Gly His Pro Thr Asn Asn ThrGln 250 255 260 gat gct tta gaa ttc tta caa aaa gcc ccc gta gac agt ctcctg aag 869 Asp Ala Leu Glu Phe Leu Gln Lys Ala Pro Val Asp Ser Leu LeuLys 265 270 275 280 aaa atg cca gct gaa aca gaa ggt gaa ata ata gaa gagttt gtc ttc 917 Lys Met Pro Ala Glu Thr Glu Gly Glu Ile Ile Glu Glu PheVal Phe 285 290 295 gta cca tca att gaa aaa gtt ttc cca tcc cac caa cctttc ttg gaa 965 Val Pro Ser Ile Glu Lys Val Phe Pro Ser His Gln Pro PheLeu Glu 300 305 310 gaa tca cca ttg gcc aga atg aaa tcc gga tcc ttt aacaaa gta cct 1013 Glu Ser Pro Leu Ala Arg Met Lys Ser Gly Ser Phe Asn LysVal Pro 315 320 325 tta tta gtt gga ttt aac agt gca gaa gga ctt ttg ttcaaa ttc ttc 1061 Leu Leu Val Gly Phe Asn Ser Ala Glu Gly Leu Leu Phe LysPhe Phe 330 335 340 atg aaa gaa aaa cca gag atg ctg aac caa gct gaa gcagat ttt gaa 1109 Met Lys Glu Lys Pro Glu Met Leu Asn Gln Ala Glu Ala AspPhe Glu 345 350 355 360 aga ctc gta cca gcc gaa ttt gaa tta gtc cat ggatca gag gaa tcg 1157 Arg Leu Val Pro Ala Glu Phe Glu Leu Val His Gly SerGlu Glu Ser 365 370 375 aaa aaa ctt gca gaa aaa atc agg aag ttt tac tttgac gat aaa ccc 1205 Lys Lys Leu Ala Glu Lys Ile Arg Lys Phe Tyr Phe AspAsp Lys Pro 380 385 390 gtt cca gaa aat gaa cag aaa ttt att gac ttg atagga gat att tgg 1253 Val Pro Glu Asn Glu Gln Lys Phe Ile Asp Leu Ile GlyAsp Ile Trp 395 400 405 ttt act aga ggt gtt gac aag cat gtc aag ttg tctgtg gag aaa caa 1301 Phe Thr Arg Gly Val Asp Lys His Val Lys Leu Ser ValGlu Lys Gln 410 415 420 gac gaa cca gtt tat tat tat gaa tat tcc ttc tcggaa agt cat cct 1349 Asp Glu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser GluSer His Pro 425 430 435 440 gca aaa gga aca ttt ggt gat cat aat ctg actggt gca tgc cat gga 1397 Ala Lys Gly Thr Phe Gly Asp His Asn Leu Thr GlyAla Cys His Gly 445 450 455 gaa gaa ctt gtg aat tta ttc aaa gtc gag atgatg aag ctg gaa aaa 1445 Glu Glu Leu Val Asn Leu Phe Lys Val Glu Met MetLys Leu Glu Lys 460 465 470 gat aaa cct aat gtt cta tta aca aaa gat agagta ctt gcc atg tgg 1493 Asp Lys Pro Asn Val Leu Leu Thr Lys Asp Arg ValLeu Ala Met Trp 475 480 485 act aac ttc atc aaa aat gga aat cct act cctgaa gta aca gaa tta 1541 Thr Asn Phe Ile Lys Asn Gly Asn Pro Thr Pro GluVal Thr Glu Leu 490 495 500 ttg cca gtt aaa tgg gaa cct gcc aca aaa gacaag ttg aat tat ttg 1589 Leu Pro Val Lys Trp Glu Pro Ala Thr Lys Asp LysLeu Asn Tyr Leu 505 510 515 520 aac att gat gcc acc tta act ttg gga acaaat cct gag gca aac cga 1637 Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr AsnPro Glu Ala Asn Arg 525 530 535 gtc aaa ttt tgg gaa gac gcc aca aaa tctttg cac ggt caa taa 1682 Val Lys Phe Trp Glu Asp Ala Thr Lys Ser Leu HisGly Gln 540 545 550 taatttatga aaattgtttt aaatacttta ggtaatatattaggtaaata aaaattaaaa 1742 aataacaatt tttatgtttt atgtattggc ttatgtgtatcagttctaat tttatttatt 1802 tattcttgtt ttgcttgttt tgaaatatca tggttttaattttcaaaaca caacgtcgtt 1862 tgtttttagc aaaatttcca atagatatgt tatattaagtactctgaagt atttttatat 1922 atacactaaa atcagtaaaa atacattaac taaaaatataagatattttc aataattttt 1982 tttaaagaaa ataccaaaaa taaagtaaaa ttccaaacggaatttttgtt taacttaaaa 2042 ataaaattaa ctcttcaata attttgataa ttagtatttctgatatcatt agtgaaaatt 2102 atattttgat aatacgtatt tatatttaaa ataaaattatgt 2144 58 550 PRT Ctenocephalides felis misc_feature (145)..(145) The′Xaa′ at location 145 stands for Asp, or Asn. 58 Met Ser Arg Val Ile PheLeu Ser Cys Ile Phe Leu Phe Ser Phe Asn 1 5 10 15 Phe Ile Lys Cys AspSer Pro Thr Val Thr Leu Pro Gln Gly Glu Leu 20 25 30 Val Gly Lys Ala LeuThr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr 35 40 45 Thr Gly Val Pro TyrAla Lys Pro Pro Val Gly Glu Leu Arg Phe Lys 50 55 60 Pro Pro Gln Lys AlaGlu Pro Trp Gln Gly Val Phe Asn Ala Thr Leu 65 70 75 80 Tyr Gly Asn ValCys Lys Ser Leu Asn Phe Phe Leu Lys Lys Ile Glu 85 90 95 Gly Asp Glu AspCys Leu Val Val Asn Val Tyr Ala Pro Lys Thr Thr 100 105 110 Ser Asp LysLys Leu Pro Val Phe Phe Trp Val His Gly Gly Gly Phe 115 120 125 Val ThrGly Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu Val 130 135 140 XaaPhe Asp Val Ile Phe Val Thr Phe Asn Tyr Arg Leu Gly Pro Leu 145 150 155160 Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu 165170 175 Leu Asp Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys180 185 190 Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly Val Ser AlaGly 195 200 205 Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr Thr ThrGly Leu 210 215 220 Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe AsnPro Trp Ala 225 230 235 240 Phe Gln Arg His Pro Val Lys Arg Ser Leu GlnLeu Ala Glu Ile Leu 245 250 255 Gly His Pro Thr Asn Asn Thr Gln Asp AlaLeu Glu Phe Leu Gln Lys 260 265 270 Ala Pro Val Asp Ser Leu Leu Lys LysMet Pro Ala Glu Thr Glu Gly 275 280 285 Glu Ile Ile Glu Glu Phe Val PheVal Pro Ser Ile Glu Lys Val Phe 290 295 300 Pro Ser His Gln Pro Phe LeuGlu Glu Ser Pro Leu Ala Arg Met Lys 305 310 315 320 Ser Gly Ser Phe AsnLys Val Pro Leu Leu Val Gly Phe Asn Ser Ala 325 330 335 Glu Gly Leu LeuPhe Lys Phe Phe Met Lys Glu Lys Pro Glu Met Leu 340 345 350 Asn Gln AlaGlu Ala Asp Phe Glu Arg Leu Val Pro Ala Glu Phe Glu 355 360 365 Leu ValHis Gly Ser Glu Glu Ser Lys Lys Leu Ala Glu Lys Ile Arg 370 375 380 LysPhe Tyr Phe Asp Asp Lys Pro Val Pro Glu Asn Glu Gln Lys Phe 385 390 395400 Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr Arg Gly Val Asp Lys His 405410 415 Val Lys Leu Ser Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu420 425 430 Tyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr Phe Gly AspHis 435 440 445 Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val Asn LeuPhe Lys 450 455 460 Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn ValLeu Leu Thr 465 470 475 480 Lys Asp Arg Val Leu Ala Met Trp Thr Asn PheIle Lys Asn Gly Asn 485 490 495 Pro Thr Pro Glu Val Thr Glu Leu Leu ProVal Lys Trp Glu Pro Ala 500 505 510 Thr Lys Asp Lys Leu Asn Tyr Leu AsnIle Asp Ala Thr Leu Thr Leu 515 520 525 Gly Thr Asn Pro Glu Ala Asn ArgVal Lys Phe Trp Glu Asp Ala Thr 530 535 540 Lys Ser Leu His Gly Gln 545550 59 2144 DNA Ctenocephalides felis 59 acataatttt attttaaatataaatacgta ttatcaaaat ataattttca ctaatgatat 60 cagaaatact aattatcaaaattattgaag agttaatttt atttttaagt taaacaaaaa 120 ttccgtttgg aattttactttatttttggt attttcttta aaaaaaatta ttgaaaatat 180 cttatatttt tagttaatgtatttttactg attttagtgt atatataaaa atacttcaga 240 gtacttaata taacatatctattggaaatt ttgctaaaaa caaacgacgt tgtgttttga 300 aaattaaaac catgatatttcaaaacaagc aaaacaagaa taaataaata aaattagaac 360 tgatacacat aagccaatacataaaacata aaaattgtta ttttttaatt tttatttacc 420 taatatatta cctaaagtatttaaaacaat tttcataaat tattattgac cgtgcaaaga 480 ttttgtggcg tcttcccaaaatttgactcg gtttgcctca ggatttgttc ccaaagttaa 540 ggtggcatca atgttcaaataattcaactt gtcttttgtg gcaggttccc atttaactgg 600 caataattct gttacttcaggagtaggatt tccatttttg atgaagttag tccacatggc 660 aagtactcta tcttttgttaatagaacatt aggtttatct ttttccagct tcatcatctc 720 gactttgaat aaattcacaagttcttctcc atggcatgca ccagtcagat tatgatcacc 780 aaatgttcct tttgcaggatgactttccga gaaggaatat tcataataat aaactggttc 840 gtcttgtttc tccacagacaacttgacatg cttgtcaaca cctctagtaa accaaatatc 900 tcctatcaag tcaataaatttctgttcatt ttctggaacg ggtttatcgt caaagtaaaa 960 cttcctgatt ttttctgcaagttttttcga ttcctctgat ccatggacta attcaaattc 1020 ggctggtacg agtctttcaaaatctgcttc agcttggttc agcatctctg gtttttcttt 1080 catgaagaat ttgaacaaaagtccttctgc actgttaaat ccaactaata aaggtacttt 1140 gttaaaggat ccggatttcattctggccaa tggtgattct tccaagaaag gttggtggga 1200 tgggaaaact ttttcaattgatggtacgaa gacaaactct tctattattt caccttctgt 1260 ttcagctggc attttcttcaggagactgtc tacgggggct ttttgtaaga attctaaagc 1320 atcttgagta ttgtttgtgggatgacccaa tatctcagca agttgaagac tacgctttac 1380 tggatgtctt tggaaggcccatggattaaa agcacttcca ctttgagcaa ttgccctttt 1440 gtaaagtcca gtggttgtatgagataacaa aagataatga acacttgctc caccagcaga 1500 aacaccacca attgtaatattttctggatc tccaccaaat ttctcaatgt tttctttggt 1560 ccatttcaga gctgccacctgatccaataa tcctacattt cctggagcac cctccaactc 1620 caaattcaga aatccgagaggtcccaatcg gtaattgaaa gttacgaaaa taacatcaaa 1680 atytactaaa taatctgggctttggaattc taaatttccg gatccagtca caaaaccacc 1740 accatgaacc cagaaaaatactggaagttt tttatcagaa gttgtttttg gtgcgtacac 1800 gtttactacc aagcagtcttcgtctccttc aattttcttc aagaagaaat ttaaagattt 1860 acacacattt ccgtataatgtggcgttgaa aacaccttgc catggctcag ctttctgtgg 1920 aggcttaaat ctaagttctccaacaggagg tttagcataa ggtacacctg tgtagctaaa 1980 atactctttt ccattttcgttcgtcaaagc ttttccaacc aattcgcctt ggggcaaagt 2040 tacagtcggg gaatcacattttataaaatt aaaactaaac aaaaaaatac aacttaaaaa 2100 aataacacga gacatcttggatctagacta ttgactatgt gtac 2144 60 1650 DNA Ctenocephalides felis exon(1)..(1650) 60 atg tct cgt gtt att ttt tta agt tgt att ttt ttg ttt agtttt aat 48 Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser PheAsn 1 5 10 15 ttt ata aaa tgt gat tcc ccg act gta act ttg ccc caa ggcgaa ttg 96 Phe Ile Lys Cys Asp Ser Pro Thr Val Thr Leu Pro Gln Gly GluLeu 20 25 30 gtt gga aaa gct ttg acg aac gaa aat gga aaa gag tat ttt agctac 144 Val Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr35 40 45 aca ggt gta cct tat gct aaa cct cct gtt gga gaa ctt aga ttt aag192 Thr Gly Val Pro Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys 5055 60 cct cca cag aaa gct gag cca tgg caa ggt gtt ttc aac gcc aca tta240 Pro Pro Gln Lys Ala Glu Pro Trp Gln Gly Val Phe Asn Ala Thr Leu 6570 75 80 tac gga aat gtg tgt aaa tct tta aat ttc ttc ttg aag aaa att gaa288 Tyr Gly Asn Val Cys Lys Ser Leu Asn Phe Phe Leu Lys Lys Ile Glu 8590 95 gga gac gaa gac tgc ttg gta gta aac gtg tac gca cca aaa aca act336 Gly Asp Glu Asp Cys Leu Val Val Asn Val Tyr Ala Pro Lys Thr Thr 100105 110 tct gat aaa aaa ctt cca gta ttt ttc tgg gtt cat ggt ggt ggt ttt384 Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His Gly Gly Gly Phe 115120 125 gtg act gga tcc gga aat tta gaa ttc caa agc cca gat tat tta gta432 Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu Val 130135 140 rat ttt gat gtt att ttc gta act ttc aat tac cga ttg gga cct ctc480 Xaa Phe Asp Val Ile Phe Val Thr Phe Asn Tyr Arg Leu Gly Pro Leu 145150 155 160 gga ttt ctg aat ttg gag ttg gag ggt gct cca gga aat gta ggatta 528 Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu165 170 175 ttg gat cag gtg gca gct ctg aaa tgg acc aaa gaa aac att gagaaa 576 Leu Asp Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys180 185 190 ttt ggt gga gat cca gaa aat att aca att ggt ggt gtt tct gctggt 624 Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly195 200 205 gga gca agt gtt cat tat ctt ttg tta tct cat aca acc act ggactt 672 Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu210 215 220 tac aaa agg gca att gct caa agt gga agt gct ttt aat cca tgggcc 720 Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala225 230 235 240 ttc caa aga cat cca gta aag cgt agt ctt caa ctt gct gagata ttg 768 Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala Glu IleLeu 245 250 255 ggt cat ccc aca aac aat act caa gat gct tta gaa ttc ttacaa aaa 816 Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu GlnLys 260 265 270 gcc ccc gta gac agt ctc ctg aag aaa atg cca gct gaa acagaa ggt 864 Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro Ala Glu Thr GluGly 275 280 285 gaa ata ata gaa gag ttt gtc ttc gta cca tca att gaa aaagtt ttc 912 Glu Ile Ile Glu Glu Phe Val Phe Val Pro Ser Ile Glu Lys ValPhe 290 295 300 cca tcc cac caa cct ttc ttg gaa gaa tca cca ttg gcc agaatg aaa 960 Pro Ser His Gln Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg MetLys 305 310 315 320 tcc gga tcc ttt aac aaa gta cct tta tta gtt gga tttaac agt gca 1008 Ser Gly Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe AsnSer Ala 325 330 335 gaa gga ctt ttg ttc aaa ttc ttc atg aaa gaa aaa ccagag atg ctg 1056 Glu Gly Leu Leu Phe Lys Phe Phe Met Lys Glu Lys Pro GluMet Leu 340 345 350 aac caa gct gaa gca gat ttt gaa aga ctc gta cca gccgaa ttt gaa 1104 Asn Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro Ala GluPhe Glu 355 360 365 tta gtc cat gga tca gag gaa tcg aaa aaa ctt gca gaaaaa atc agg 1152 Leu Val His Gly Ser Glu Glu Ser Lys Lys Leu Ala Glu LysIle Arg 370 375 380 aag ttt tac ttt gac gat aaa ccc gtt cca gaa aat gaacag aaa ttt 1200 Lys Phe Tyr Phe Asp Asp Lys Pro Val Pro Glu Asn Glu GlnLys Phe 385 390 395 400 att gac ttg ata gga gat att tgg ttt act aga ggtgtt gac aag cat 1248 Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr Arg Gly ValAsp Lys His 405 410 415 gtc aag ttg tct gtg gag aaa caa gac gaa cca gtttat tat tat gaa 1296 Val Lys Leu Ser Val Glu Lys Gln Asp Glu Pro Val TyrTyr Tyr Glu 420 425 430 tat tcc ttc tcg gaa agt cat cct gca aaa gga acattt ggt gat cat 1344 Tyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr PheGly Asp His 435 440 445 aat ctg act ggt gca tgc cat gga gaa gaa ctt gtgaat tta ttc aaa 1392 Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val AsnLeu Phe Lys 450 455 460 gtc gag atg atg aag ctg gaa aaa gat aaa cct aatgtt cta tta aca 1440 Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn ValLeu Leu Thr 465 470 475 480 aaa gat aga gta ctt gcc atg tgg act aac ttcatc aaa aat gga aat 1488 Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe IleLys Asn Gly Asn 485 490 495 cct act cct gaa gta aca gaa tta ttg cca gttaaa tgg gaa cct gcc 1536 Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val LysTrp Glu Pro Ala 500 505 510 aca aaa gac aag ttg aat tat ttg aac att gatgcc acc tta act ttg 1584 Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp AlaThr Leu Thr Leu 515 520 525 gga aca aat cct gag gca aac cga gtc aaa ttttgg gaa gac gcc aca 1632 Gly Thr Asn Pro Glu Ala Asn Arg Val Lys Phe TrpGlu Asp Ala Thr 530 535 540 aaa tct ttg cac ggt caa 1650 Lys Ser Leu HisGly Gln 545 550 61 1650 DNA Ctenocephalides felis 61 ttgaccgtgcaaagattttg tggcgtcttc ccaaaatttg actcggtttg cctcaggatt 60 tgttcccaaagttaaggtgg catcaatgtt caaataattc aacttgtctt ttgtggcagg 120 ttcccatttaactggcaata attctgttac ttcaggagta ggatttccat ttttgatgaa 180 gttagtccacatggcaagta ctctatcttt tgttaataga acattaggtt tatctttttc 240 cagcttcatcatctcgactt tgaataaatt cacaagttct tctccatggc atgcaccagt 300 cagattatgatcaccaaatg ttccttttgc aggatgactt tccgagaagg aatattcata 360 ataataaactggttcgtctt gtttctccac agacaacttg acatgcttgt caacacctct 420 agtaaaccaaatatctccta tcaagtcaat aaatttctgt tcattttctg gaacgggttt 480 atcgtcaaagtaaaacttcc tgattttttc tgcaagtttt ttcgattcct ctgatccatg 540 gactaattcaaattcggctg gtacgagtct ttcaaaatct gcttcagctt ggttcagcat 600 ctctggtttttctttcatga agaatttgaa caaaagtcct tctgcactgt taaatccaac 660 taataaaggtactttgttaa aggatccgga tttcattctg gccaatggtg attcttccaa 720 gaaaggttggtgggatggga aaactttttc aattgatggt acgaagacaa actcttctat 780 tatttcaccttctgtttcag ctggcatttt cttcaggaga ctgtctacgg gggctttttg 840 taagaattctaaagcatctt gagtattgtt tgtgggatga cccaatatct cagcaagttg 900 aagactacgctttactggat gtctttggaa ggcccatgga ttaaaagcac ttccactttg 960 agcaattgcccttttgtaaa gtccagtggt tgtatgagat aacaaaagat aatgaacact 1020 tgctccaccagcagaaacac caccaattgt aatattttct ggatctccac caaatttctc 1080 aatgttttctttggtccatt tcagagctgc cacctgatcc aataatccta catttcctgg 1140 agcaccctccaactccaaat tcagaaatcc gagaggtccc aatcggtaat tgaaagttac 1200 gaaaataacatcaaaatyta ctaaataatc tgggctttgg aattctaaat ttccggatcc 1260 agtcacaaaaccaccaccat gaacccagaa aaatactgga agttttttat cagaagttgt 1320 ttttggtgcgtacacgttta ctaccaagca gtcttcgtct ccttcaattt tcttcaagaa 1380 gaaatttaaagatttacaca catttccgta taatgtggcg ttgaaaacac cttgccatgg 1440 ctcagctttctgtggaggct taaatctaag ttctccaaca ggaggtttag cataaggtac 1500 acctgtgtagctaaaatact cttttccatt ttcgttcgtc aaagcttttc caaccaattc 1560 gccttggggcaaagttacag tcggggaatc acattttata aaattaaaac taaacaaaaa 1620 aatacaacttaaaaaaataa cacgagacat 1650 62 29 DNA Artificial sequence SyntheticPrimer 62 aaactcgagt cccccgactg taactttgc 29 63 36 DNA Artificialsequence Synthetic Primer 63 tcatctgcag ttattgactg tgcaaagttt ttgtgg 3664 32 DNA Artificial sequence Synthetic Primer 64 ttccggatcc ggctgatctacaagtgactt tg 32 65 34 DNA Artificial sequence Synthetic Primer 65tggtactcga gtcataaaaa tttattccaa aatc 34 66 39 DNA Artificial sequenceSynthetic Primer 66 aaaactgcag tataaatatg ttacctcaca gtgcattag 39 671987 DNA Ctenocephalides felis CDS (231)..(1820) 67 aattcacagtgtaaataatt ttatttgata taaatgtatt taatttttat tttaatctaa 60 ttttaatttaaatatatata gttttattta taaaaaaata ttttttttat gatcgaaaag 120 aaatttttatttatgtttat gagtgtgtgt tttggctatg atttacatta tttttgagct 180 agtataaaattaaaccatat tatattttgg atatataata acattttata atg tgt 236 Met Cys 1 gatcca tta cta aaa aca aca aca tat gga att ctg aaa ggc aag aaa 284 Asp ProLeu Leu Lys Thr Thr Thr Tyr Gly Ile Leu Lys Gly Lys Lys 5 10 15 gtt gtaaac gaa aat ggt aaa att tac tat agt tac aca ggt ata ccc 332 Val Val AsnGlu Asn Gly Lys Ile Tyr Tyr Ser Tyr Thr Gly Ile Pro 20 25 30 tat gca aaatct cct gta aat gat ctc aga ttc aag cca cca caa aaa 380 Tyr Ala Lys SerPro Val Asn Asp Leu Arg Phe Lys Pro Pro Gln Lys 35 40 45 50 ctt gat ccttgg aat ggt gtt ttt gac gcc act cag tat gga aat aat 428 Leu Asp Pro TrpAsn Gly Val Phe Asp Ala Thr Gln Tyr Gly Asn Asn 55 60 65 tgt gct gct gggaaa tgg ttt ttg aaa tca gct ggg ggt tgc gaa gat 476 Cys Ala Ala Gly LysTrp Phe Leu Lys Ser Ala Gly Gly Cys Glu Asp 70 75 80 tgc ctt tac tta aatatc tat gtc cca caa aac act tca gaa aat cct 524 Cys Leu Tyr Leu Asn IleTyr Val Pro Gln Asn Thr Ser Glu Asn Pro 85 90 95 ttg cca gta atg ttt tggatt cat gga gga gca ttt gtg gtc gga tca 572 Leu Pro Val Met Phe Trp IleHis Gly Gly Ala Phe Val Val Gly Ser 100 105 110 gga aat tct gat ata catggt cct gat tat tta ata gaa tat gat att 620 Gly Asn Ser Asp Ile His GlyPro Asp Tyr Leu Ile Glu Tyr Asp Ile 115 120 125 130 atc tta gta act attaat tat cgt cta gga cca ctt ggt ttt ctt aat 668 Ile Leu Val Thr Ile AsnTyr Arg Leu Gly Pro Leu Gly Phe Leu Asn 135 140 145 ttg gaa atc gaa gatgcg cct ggg aat gtt gga ttg atg gat caa gtt 716 Leu Glu Ile Glu Asp AlaPro Gly Asn Val Gly Leu Met Asp Gln Val 150 155 160 gca gcc cta aaa tgggta aat gaa aat att gca acc ttt agt gga gac 764 Ala Ala Leu Lys Trp ValAsn Glu Asn Ile Ala Thr Phe Ser Gly Asp 165 170 175 cca aaa aat att acaatt tgt gga gca act gct gga gct gca agt gta 812 Pro Lys Asn Ile Thr IleCys Gly Ala Thr Ala Gly Ala Ala Ser Val 180 185 190 cat tat cac att ttgtca caa ctt acc aaa ggt tta ttc cac aag gct 860 His Tyr His Ile Leu SerGln Leu Thr Lys Gly Leu Phe His Lys Ala 195 200 205 210 ata gca caa agtgga agt gct ttt aat ccc tgg gct ttc caa aaa aat 908 Ile Ala Gln Ser GlySer Ala Phe Asn Pro Trp Ala Phe Gln Lys Asn 215 220 225 cct gtt aag aatgca ctt cga cta tgc aaa acc tta ggc ctt acc aca 956 Pro Val Lys Asn AlaLeu Arg Leu Cys Lys Thr Leu Gly Leu Thr Thr 230 235 240 aac aac ctt caagaa gcc ttg gat ttt ttg aaa aac cta cca gta gaa 1004 Asn Asn Leu Gln GluAla Leu Asp Phe Leu Lys Asn Leu Pro Val Glu 245 250 255 aca ttg tta aatacc aaa tta ccc caa gaa att gat ggt caa ctg ctg 1052 Thr Leu Leu Asn ThrLys Leu Pro Gln Glu Ile Asp Gly Gln Leu Leu 260 265 270 gat gac ttc gtgttt gta cct tcg att gaa aaa aca ttt cca gaa caa 1100 Asp Asp Phe Val PheVal Pro Ser Ile Glu Lys Thr Phe Pro Glu Gln 275 280 285 290 gat tcg tactta act gac ttg cca ata cca ata ata aat tca gga aaa 1148 Asp Ser Tyr LeuThr Asp Leu Pro Ile Pro Ile Ile Asn Ser Gly Lys 295 300 305 ttc cac aaagtt cca ttg ttg aca ggt tac aac agt gcc gaa ggc aat 1196 Phe His Lys ValPro Leu Leu Thr Gly Tyr Asn Ser Ala Glu Gly Asn 310 315 320 cta ttt ttcatg tac tta aaa aca gat cca gat tta tta aat aaa ttt 1244 Leu Phe Phe MetTyr Leu Lys Thr Asp Pro Asp Leu Leu Asn Lys Phe 325 330 335 gaa gct gatttt gaa aga ttt ata cca act gac tta gaa tta cct ttg 1292 Glu Ala Asp PheGlu Arg Phe Ile Pro Thr Asp Leu Glu Leu Pro Leu 340 345 350 cga tca caaaaa tct att gca ctg ggt gaa gca atc agg gaa ttt tat 1340 Arg Ser Gln LysSer Ile Ala Leu Gly Glu Ala Ile Arg Glu Phe Tyr 355 360 365 370 ttc caaaac aaa acc ata tca gaa aat atg cag aat ttt gta gat gtt 1388 Phe Gln AsnLys Thr Ile Ser Glu Asn Met Gln Asn Phe Val Asp Val 375 380 385 tta agtgat aat tgg ttt aca cgt gga att gat gag caa gta aag tta 1436 Leu Ser AspAsn Trp Phe Thr Arg Gly Ile Asp Glu Gln Val Lys Leu 390 395 400 act gttaaa aat cag gaa gaa cca gtt ttt tat tat gtt tat aat ttt 1484 Thr Val LysAsn Gln Glu Glu Pro Val Phe Tyr Tyr Val Tyr Asn Phe 405 410 415 gat gaaaat tct cca agt cgg aaa gtt ttt ggt gat ttt gga ata aaa 1532 Asp Glu AsnSer Pro Ser Arg Lys Val Phe Gly Asp Phe Gly Ile Lys 420 425 430 ggc ggtggt cat gct gat gaa ttg ggt aat ata ttt aaa gcc aaa agt 1580 Gly Gly GlyHis Ala Asp Glu Leu Gly Asn Ile Phe Lys Ala Lys Ser 435 440 445 450 gcaaat ttt ggg aag gaa aca cca aat gct gtg ttg gtt cag aga agg 1628 Ala AsnPhe Gly Lys Glu Thr Pro Asn Ala Val Leu Val Gln Arg Arg 455 460 465 atgctg gag atg tgg act aat ttt gct aaa ttt gga aat cct act cca 1676 Met LeuGlu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn Pro Thr Pro 470 475 480 gctatt acg gat aca ctt cca ata aaa tgg gaa cct gct ttt aaa gaa 1724 Ala IleThr Asp Thr Leu Pro Ile Lys Trp Glu Pro Ala Phe Lys Glu 485 490 495 aatatg act ttt gtt caa att gac att gat tta aat ttg agt act gat 1772 Asn MetThr Phe Val Gln Ile Asp Ile Asp Leu Asn Leu Ser Thr Asp 500 505 510 ccacta aaa agt cgt atg gaa ttt ggg aat aaa ata aaa tta tta aaa 1820 Pro LeuLys Ser Arg Met Glu Phe Gly Asn Lys Ile Lys Leu Leu Lys 515 520 525 530taagtaacta tacttagcta aaccataata taccaaataa tagtatagga atacttcaca 1880attttttgtt acttcgttaa gtaaatttaa ttttttataa aaccaacttt tacgaataaa 1940aaatgtaatt attttggaaa aaaaaaagaa aaaaaaaaaa aaaaaac 1987 68 530 PRTCtenocephalides felis 68 Met Cys Asp Pro Leu Leu Lys Thr Thr Thr Tyr GlyIle Leu Lys Gly 1 5 10 15 Lys Lys Val Val Asn Glu Asn Gly Lys Ile TyrTyr Ser Tyr Thr Gly 20 25 30 Ile Pro Tyr Ala Lys Ser Pro Val Asn Asp LeuArg Phe Lys Pro Pro 35 40 45 Gln Lys Leu Asp Pro Trp Asn Gly Val Phe AspAla Thr Gln Tyr Gly 50 55 60 Asn Asn Cys Ala Ala Gly Lys Trp Phe Leu LysSer Ala Gly Gly Cys 65 70 75 80 Glu Asp Cys Leu Tyr Leu Asn Ile Tyr ValPro Gln Asn Thr Ser Glu 85 90 95 Asn Pro Leu Pro Val Met Phe Trp Ile HisGly Gly Ala Phe Val Val 100 105 110 Gly Ser Gly Asn Ser Asp Ile His GlyPro Asp Tyr Leu Ile Glu Tyr 115 120 125 Asp Ile Ile Leu Val Thr Ile AsnTyr Arg Leu Gly Pro Leu Gly Phe 130 135 140 Leu Asn Leu Glu Ile Glu AspAla Pro Gly Asn Val Gly Leu Met Asp 145 150 155 160 Gln Val Ala Ala LeuLys Trp Val Asn Glu Asn Ile Ala Thr Phe Ser 165 170 175 Gly Asp Pro LysAsn Ile Thr Ile Cys Gly Ala Thr Ala Gly Ala Ala 180 185 190 Ser Val HisTyr His Ile Leu Ser Gln Leu Thr Lys Gly Leu Phe His 195 200 205 Lys AlaIle Ala Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln 210 215 220 LysAsn Pro Val Lys Asn Ala Leu Arg Leu Cys Lys Thr Leu Gly Leu 225 230 235240 Thr Thr Asn Asn Leu Gln Glu Ala Leu Asp Phe Leu Lys Asn Leu Pro 245250 255 Val Glu Thr Leu Leu Asn Thr Lys Leu Pro Gln Glu Ile Asp Gly Gln260 265 270 Leu Leu Asp Asp Phe Val Phe Val Pro Ser Ile Glu Lys Thr PhePro 275 280 285 Glu Gln Asp Ser Tyr Leu Thr Asp Leu Pro Ile Pro Ile IleAsn Ser 290 295 300 Gly Lys Phe His Lys Val Pro Leu Leu Thr Gly Tyr AsnSer Ala Glu 305 310 315 320 Gly Asn Leu Phe Phe Met Tyr Leu Lys Thr AspPro Asp Leu Leu Asn 325 330 335 Lys Phe Glu Ala Asp Phe Glu Arg Phe IlePro Thr Asp Leu Glu Leu 340 345 350 Pro Leu Arg Ser Gln Lys Ser Ile AlaLeu Gly Glu Ala Ile Arg Glu 355 360 365 Phe Tyr Phe Gln Asn Lys Thr IleSer Glu Asn Met Gln Asn Phe Val 370 375 380 Asp Val Leu Ser Asp Asn TrpPhe Thr Arg Gly Ile Asp Glu Gln Val 385 390 395 400 Lys Leu Thr Val LysAsn Gln Glu Glu Pro Val Phe Tyr Tyr Val Tyr 405 410 415 Asn Phe Asp GluAsn Ser Pro Ser Arg Lys Val Phe Gly Asp Phe Gly 420 425 430 Ile Lys GlyGly Gly His Ala Asp Glu Leu Gly Asn Ile Phe Lys Ala 435 440 445 Lys SerAla Asn Phe Gly Lys Glu Thr Pro Asn Ala Val Leu Val Gln 450 455 460 ArgArg Met Leu Glu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn Pro 465 470 475480 Thr Pro Ala Ile Thr Asp Thr Leu Pro Ile Lys Trp Glu Pro Ala Phe 485490 495 Lys Glu Asn Met Thr Phe Val Gln Ile Asp Ile Asp Leu Asn Leu Ser500 505 510 Thr Asp Pro Leu Lys Ser Arg Met Glu Phe Gly Asn Lys Ile LysLeu 515 520 525 Leu Lys 530 69 1987 DNA Ctenocephalides felis 69gttttttttt tttttttttc tttttttttt ccaaaataat tacatttttt attcgtaaaa 60gttggtttta taaaaaatta aatttactta acgaagtaac aaaaaattgt gaagtattcc 120tatactatta tttggtatat tatggtttag ctaagtatag ttacttattt taataatttt 180attttattcc caaattccat acgacttttt agtggatcag tactcaaatt taaatcaatg 240tcaatttgaa caaaagtcat attttcttta aaagcaggtt cccattttat tggaagtgta 300tccgtaatag ctggagtagg atttccaaat ttagcaaaat tagtccacat ctccagcatc 360cttctctgaa ccaacacagc atttggtgtt tccttcccaa aatttgcact tttggcttta 420aatatattac ccaattcatc agcatgacca ccgcctttta ttccaaaatc accaaaaact 480ttccgacttg gagaattttc atcaaaatta taaacataat aaaaaactgg ttcttcctga 540tttttaacag ttaactttac ttgctcatca attccacgtg taaaccaatt atcacttaaa 600acatctacaa aattctgcat attttctgat atggttttgt tttggaaata aaattccctg 660attgcttcac ccagtgcaat agatttttgt gatcgcaaag gtaattctaa gtcagttggt 720ataaatcttt caaaatcagc ttcaaattta tttaataaat ctggatctgt ttttaagtac 780atgaaaaata gattgccttc ggcactgttg taacctgtca acaatggaac tttgtggaat 840tttcctgaat ttattattgg tattggcaag tcagttaagt acgaatcttg ttctggaaat 900gttttttcaa tcgaaggtac aaacacgaag tcatccagca gttgaccatc aatttcttgg 960ggtaatttgg tatttaacaa tgtttctact ggtaggtttt tcaaaaaatc caaggcttct 1020tgaaggttgt ttgtggtaag gcctaaggtt ttgcatagtc gaagtgcatt cttaacagga 1080tttttttgga aagcccaggg attaaaagca cttccacttt gtgctatagc cttgtggaat 1140aaacctttgg taagttgtga caaaatgtga taatgtacac ttgcagctcc agcagttgct 1200ccacaaattg taatattttt tgggtctcca ctaaaggttg caatattttc atttacccat 1260tttagggctg caacttgatc catcaatcca acattcccag gcgcatcttc gatttccaaa 1320ttaagaaaac caagtggtcc tagacgataa ttaatagtta ctaagataat atcatattct 1380attaaataat caggaccatg tatatcagaa tttcctgatc cgaccacaaa tgctcctcca 1440tgaatccaaa acattactgg caaaggattt tctgaagtgt tttgtgggac atagatattt 1500aagtaaaggc aatcttcgca acccccagct gatttcaaaa accatttccc agcagcacaa 1560ttatttccat actgagtggc gtcaaaaaca ccattccaag gatcaagttt ttgtggtggc 1620ttgaatctga gatcatttac aggagatttt gcatagggta tacctgtgta actatagtaa 1680attttaccat tttcgtttac aactttcttg cctttcagaa ttccatatgt tgttgttttt 1740agtaatggat cacacattat aaaatgttat tatatatcca aaatataata tggtttaatt 1800ttatactagc tcaaaaataa tgtaaatcat agccaaaaca cacactcata aacataaata 1860aaaatttctt ttcgatcata aaaaaaatat ttttttataa ataaaactat atatatttaa 1920attaaaatta gattaaaata aaaattaaat acatttatat caaataaaat tatttacact 1980gtgaatt 1987 70 1590 DNA Ctenocephalides felis exon (1)..(1590) 70 atgtgt gat cca tta cta aaa aca aca aca tat gga att ctg aaa ggc 48 Met CysAsp Pro Leu Leu Lys Thr Thr Thr Tyr Gly Ile Leu Lys Gly 1 5 10 15 aagaaa gtt gta aac gaa aat ggt aaa att tac tat agt tac aca ggt 96 Lys LysVal Val Asn Glu Asn Gly Lys Ile Tyr Tyr Ser Tyr Thr Gly 20 25 30 ata ccctat gca aaa tct cct gta aat gat ctc aga ttc aag cca cca 144 Ile Pro TyrAla Lys Ser Pro Val Asn Asp Leu Arg Phe Lys Pro Pro 35 40 45 caa aaa cttgat cct tgg aat ggt gtt ttt gac gcc act cag tat gga 192 Gln Lys Leu AspPro Trp Asn Gly Val Phe Asp Ala Thr Gln Tyr Gly 50 55 60 aat aat tgt gctgct ggg aaa tgg ttt ttg aaa tca gct ggg ggt tgc 240 Asn Asn Cys Ala AlaGly Lys Trp Phe Leu Lys Ser Ala Gly Gly Cys 65 70 75 80 gaa gat tgc ctttac tta aat atc tat gtc cca caa aac act tca gaa 288 Glu Asp Cys Leu TyrLeu Asn Ile Tyr Val Pro Gln Asn Thr Ser Glu 85 90 95 aat cct ttg cca gtaatg ttt tgg att cat gga gga gca ttt gtg gtc 336 Asn Pro Leu Pro Val MetPhe Trp Ile His Gly Gly Ala Phe Val Val 100 105 110 gga tca gga aat tctgat ata cat ggt cct gat tat tta ata gaa tat 384 Gly Ser Gly Asn Ser AspIle His Gly Pro Asp Tyr Leu Ile Glu Tyr 115 120 125 gat att atc tta gtaact att aat tat cgt cta gga cca ctt ggt ttt 432 Asp Ile Ile Leu Val ThrIle Asn Tyr Arg Leu Gly Pro Leu Gly Phe 130 135 140 ctt aat ttg gaa atcgaa gat gcg cct ggg aat gtt gga ttg atg gat 480 Leu Asn Leu Glu Ile GluAsp Ala Pro Gly Asn Val Gly Leu Met Asp 145 150 155 160 caa gtt gca gcccta aaa tgg gta aat gaa aat att gca acc ttt agt 528 Gln Val Ala Ala LeuLys Trp Val Asn Glu Asn Ile Ala Thr Phe Ser 165 170 175 gga gac cca aaaaat att aca att tgt gga gca act gct gga gct gca 576 Gly Asp Pro Lys AsnIle Thr Ile Cys Gly Ala Thr Ala Gly Ala Ala 180 185 190 agt gta cat tatcac att ttg tca caa ctt acc aaa ggt tta ttc cac 624 Ser Val His Tyr HisIle Leu Ser Gln Leu Thr Lys Gly Leu Phe His 195 200 205 aag gct ata gcacaa agt gga agt gct ttt aat ccc tgg gct ttc caa 672 Lys Ala Ile Ala GlnSer Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln 210 215 220 aaa aat cct gttaag aat gca ctt cga cta tgc aaa acc tta ggc ctt 720 Lys Asn Pro Val LysAsn Ala Leu Arg Leu Cys Lys Thr Leu Gly Leu 225 230 235 240 acc aca aacaac ctt caa gaa gcc ttg gat ttt ttg aaa aac cta cca 768 Thr Thr Asn AsnLeu Gln Glu Ala Leu Asp Phe Leu Lys Asn Leu Pro 245 250 255 gta gaa acattg tta aat acc aaa tta ccc caa gaa att gat ggt caa 816 Val Glu Thr LeuLeu Asn Thr Lys Leu Pro Gln Glu Ile Asp Gly Gln 260 265 270 ctg ctg gatgac ttc gtg ttt gta cct tcg att gaa aaa aca ttt cca 864 Leu Leu Asp AspPhe Val Phe Val Pro Ser Ile Glu Lys Thr Phe Pro 275 280 285 gaa caa gattcg tac tta act gac ttg cca ata cca ata ata aat tca 912 Glu Gln Asp SerTyr Leu Thr Asp Leu Pro Ile Pro Ile Ile Asn Ser 290 295 300 gga aaa ttccac aaa gtt cca ttg ttg aca ggt tac aac agt gcc gaa 960 Gly Lys Phe HisLys Val Pro Leu Leu Thr Gly Tyr Asn Ser Ala Glu 305 310 315 320 ggc aatcta ttt ttc atg tac tta aaa aca gat cca gat tta tta aat 1008 Gly Asn LeuPhe Phe Met Tyr Leu Lys Thr Asp Pro Asp Leu Leu Asn 325 330 335 aaa tttgaa gct gat ttt gaa aga ttt ata cca act gac tta gaa tta 1056 Lys Phe GluAla Asp Phe Glu Arg Phe Ile Pro Thr Asp Leu Glu Leu 340 345 350 cct ttgcga tca caa aaa tct att gca ctg ggt gaa gca atc agg gaa 1104 Pro Leu ArgSer Gln Lys Ser Ile Ala Leu Gly Glu Ala Ile Arg Glu 355 360 365 ttt tatttc caa aac aaa acc ata tca gaa aat atg cag aat ttt gta 1152 Phe Tyr PheGln Asn Lys Thr Ile Ser Glu Asn Met Gln Asn Phe Val 370 375 380 gat gtttta agt gat aat tgg ttt aca cgt gga att gat gag caa gta 1200 Asp Val LeuSer Asp Asn Trp Phe Thr Arg Gly Ile Asp Glu Gln Val 385 390 395 400 aagtta act gtt aaa aat cag gaa gaa cca gtt ttt tat tat gtt tat 1248 Lys LeuThr Val Lys Asn Gln Glu Glu Pro Val Phe Tyr Tyr Val Tyr 405 410 415 aatttt gat gaa aat tct cca agt cgg aaa gtt ttt ggt gat ttt gga 1296 Asn PheAsp Glu Asn Ser Pro Ser Arg Lys Val Phe Gly Asp Phe Gly 420 425 430 ataaaa ggc ggt ggt cat gct gat gaa ttg ggt aat ata ttt aaa gcc 1344 Ile LysGly Gly Gly His Ala Asp Glu Leu Gly Asn Ile Phe Lys Ala 435 440 445 aaaagt gca aat ttt ggg aag gaa aca cca aat gct gtg ttg gtt cag 1392 Lys SerAla Asn Phe Gly Lys Glu Thr Pro Asn Ala Val Leu Val Gln 450 455 460 agaagg atg ctg gag atg tgg act aat ttt gct aaa ttt gga aat cct 1440 Arg ArgMet Leu Glu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn Pro 465 470 475 480act cca gct att acg gat aca ctt cca ata aaa tgg gaa cct gct ttt 1488 ThrPro Ala Ile Thr Asp Thr Leu Pro Ile Lys Trp Glu Pro Ala Phe 485 490 495aaa gaa aat atg act ttt gtt caa att gac att gat tta aat ttg agt 1536 LysGlu Asn Met Thr Phe Val Gln Ile Asp Ile Asp Leu Asn Leu Ser 500 505 510act gat cca cta aaa agt cgt atg gaa ttt ggg aat aaa ata aaa tta 1584 ThrAsp Pro Leu Lys Ser Arg Met Glu Phe Gly Asn Lys Ile Lys Leu 515 520 525tta aaa 1590 Leu Lys 530 71 1590 DNA Ctenocephalides felis 71 ttttaataattttattttat tcccaaattc catacgactt tttagtggat cagtactcaa 60 atttaaatcaatgtcaattt gaacaaaagt catattttct ttaaaagcag gttcccattt 120 tattggaagtgtatccgtaa tagctggagt aggatttcca aatttagcaa aattagtcca 180 catctccagcatccttctct gaaccaacac agcatttggt gtttccttcc caaaatttgc 240 acttttggctttaaatatat tacccaattc atcagcatga ccaccgcctt ttattccaaa 300 atcaccaaaaactttccgac ttggagaatt ttcatcaaaa ttataaacat aataaaaaac 360 tggttcttcctgatttttaa cagttaactt tacttgctca tcaattccac gtgtaaacca 420 attatcacttaaaacatcta caaaattctg catattttct gatatggttt tgttttggaa 480 ataaaattccctgattgctt cacccagtgc aatagatttt tgtgatcgca aaggtaattc 540 taagtcagttggtataaatc tttcaaaatc agcttcaaat ttatttaata aatctggatc 600 tgtttttaagtacatgaaaa atagattgcc ttcggcactg ttgtaacctg tcaacaatgg 660 aactttgtggaattttcctg aatttattat tggtattggc aagtcagtta agtacgaatc 720 ttgttctggaaatgtttttt caatcgaagg tacaaacacg aagtcatcca gcagttgacc 780 atcaatttcttggggtaatt tggtatttaa caatgtttct actggtaggt ttttcaaaaa 840 atccaaggcttcttgaaggt tgtttgtggt aaggcctaag gttttgcata gtcgaagtgc 900 attcttaacaggattttttt ggaaagccca gggattaaaa gcacttccac tttgtgctat 960 agccttgtggaataaacctt tggtaagttg tgacaaaatg tgataatgta cacttgcagc 1020 tccagcagttgctccacaaa ttgtaatatt ttttgggtct ccactaaagg ttgcaatatt 1080 ttcatttacccattttaggg ctgcaacttg atccatcaat ccaacattcc caggcgcatc 1140 ttcgatttccaaattaagaa aaccaagtgg tcctagacga taattaatag ttactaagat 1200 aatatcatattctattaaat aatcaggacc atgtatatca gaatttcctg atccgaccac 1260 aaatgctcctccatgaatcc aaaacattac tggcaaagga ttttctgaag tgttttgtgg 1320 gacatagatatttaagtaaa ggcaatcttc gcaaccccca gctgatttca aaaaccattt 1380 cccagcagcacaattatttc catactgagt ggcgtcaaaa acaccattcc aaggatcaag 1440 tttttgtggtggcttgaatc tgagatcatt tacaggagat tttgcatagg gtatacctgt 1500 gtaactatagtaaattttac cattttcgtt tacaactttc ttgcctttca gaattccata 1560 tgttgttgtttttagtaatg gatcacacat 1590 72 650 DNA Ctenocephalides felis CDS(3)..(650) 72 gg atc cat gga ggc gca ttc aac caa gga tca gga tct tat aatttt 47 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr Asn Phe 1 510 15 ttt gga cct gat tat ttg atc agg gaa gga att att ttg gtc act atc 95Phe Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val Thr Ile 20 25 30aac tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg gaa tgg gat 143 AsnTyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp 35 40 45 atccat gga aat atg ggt cta aaa gac cag aga ttg gca cta aaa tgg 191 Ile HisGly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp 50 55 60 gtt tacgac aac atc gaa aag ttt ggt gga gac aga gaa aaa att aca 239 Val Tyr AspAsn Ile Glu Lys Phe Gly Gly Asp Arg Glu Lys Ile Thr 65 70 75 att gct ggagaa tct gct gga gca gca agt gtc cat ttt ctg atg atg 287 Ile Ala Gly GluSer Ala Gly Ala Ala Ser Val His Phe Leu Met Met 80 85 90 95 gac aac tcgact aga aaa tac tac caa agg gcc att ttg cag agt ggg 335 Asp Asn Ser ThrArg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln Ser Gly 100 105 110 aca tta ctaaat ccg act gct aat caa att caa ctt ctg cat aga ttt 383 Thr Leu Leu AsnPro Thr Ala Asn Gln Ile Gln Leu Leu His Arg Phe 115 120 125 gaa aaa ctcaaa caa gtg cta aac atc acg caa aaa caa gaa ctc cta 431 Glu Lys Leu LysGln Val Leu Asn Ile Thr Gln Lys Gln Glu Leu Leu 130 135 140 aac ctg gataaa aac cta att tta cga gca gcc tta aac aga gtt cct 479 Asn Leu Asp LysAsn Leu Ile Leu Arg Ala Ala Leu Asn Arg Val Pro 145 150 155 gat agc aacgac cat gac cga gac aca gta cca gta ttt aat cca gtc 527 Asp Ser Asn AspHis Asp Arg Asp Thr Val Pro Val Phe Asn Pro Val 160 165 170 175 tta gaatca cca gaa tct cca gat cca ata aca ttt cca tct gcc ttg 575 Leu Glu SerPro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu 180 185 190 gaa agaatg aga aat ggt gaa ttt cct gat gtc gat gtc atc att ggt 623 Glu Arg MetArg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly 195 200 205 ttc aatagt gct gaa ggt tta aga tct 650 Phe Asn Ser Ala Glu Gly Leu Arg Ser 210215 73 216 PRT Ctenocephalides felis 73 Ile His Gly Gly Ala Phe Asn GlnGly Ser Gly Ser Tyr Asn Phe Phe 1 5 10 15 Gly Pro Asp Tyr Leu Ile ArgGlu Gly Ile Ile Leu Val Thr Ile Asn 20 25 30 Tyr Arg Leu Gly Val Phe GlyPhe Leu Ser Ala Pro Glu Trp Asp Ile 35 40 45 His Gly Asn Met Gly Leu LysAsp Gln Arg Leu Ala Leu Lys Trp Val 50 55 60 Tyr Asp Asn Ile Glu Lys PheGly Gly Asp Arg Glu Lys Ile Thr Ile 65 70 75 80 Ala Gly Glu Ser Ala GlyAla Ala Ser Val His Phe Leu Met Met Asp 85 90 95 Asn Ser Thr Arg Lys TyrTyr Gln Arg Ala Ile Leu Gln Ser Gly Thr 100 105 110 Leu Leu Asn Pro ThrAla Asn Gln Ile Gln Leu Leu His Arg Phe Glu 115 120 125 Lys Leu Lys GlnVal Leu Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn 130 135 140 Leu Asp LysAsn Leu Ile Leu Arg Ala Ala Leu Asn Arg Val Pro Asp 145 150 155 160 SerAsn Asp His Asp Arg Asp Thr Val Pro Val Phe Asn Pro Val Leu 165 170 175Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 180 185190 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 195200 205 Asn Ser Ala Glu Gly Leu Arg Ser 210 215 74 15 PRT PeptideMISC_FEATURE (3)..(3) Xaa = unknown 74 Asp Leu Xaa Val Xaa Xaa Leu GlnGly Thr Leu Lys Gly Lys Glu 1 5 10 15 75 31 DNA Artificial sequenceSynthetic Primer 75 cgcggatccg ctgatctaca agtgactttg c 31 76 1488 DNACtenocephalides felis exon (3)..(1487) 76 cc cag ggc gaa ttg gtt gga aaagct ttg acg aac gaa aat gga aaa 47 Gln Gly Glu Leu Val Gly Lys Ala LeuThr Asn Glu Asn Gly Lys 1 5 10 15 gag tat ttt agc tac aca ggt gtg ccttat gct aaa cct cca gtt gga 95 Glu Tyr Phe Ser Tyr Thr Gly Val Pro TyrAla Lys Pro Pro Val Gly 20 25 30 gaa ctt aga ttt aag cct cca cag aaa gctgag cca tgg aat ggt gtt 143 Glu Leu Arg Phe Lys Pro Pro Gln Lys Ala GluPro Trp Asn Gly Val 35 40 45 ttc aac gcc aca tca cat gga aat gtg tgc aaagct ttg aat ttc ttc 191 Phe Asn Ala Thr Ser His Gly Asn Val Cys Lys AlaLeu Asn Phe Phe 50 55 60 ttg aaa aaa att gaa gga gac gaa gac tgc ttg ttggtg aat gtg tac 239 Leu Lys Lys Ile Glu Gly Asp Glu Asp Cys Leu Leu ValAsn Val Tyr 65 70 75 gca cca aaa aca act tct gac aaa aaa ctt cca gta tttttc tgg gtt 287 Ala Pro Lys Thr Thr Ser Asp Lys Lys Leu Pro Val Phe PheTrp Val 80 85 90 95 cat ggt ggc ggt ttt gtg act gga tcc gga aat tta gaattt caa agc 335 His Gly Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu PheGln Ser 100 105 110 cca gat tat tta gta aat tat gat gtt att ttt gta actttc aat tac 383 Pro Asp Tyr Leu Val Asn Tyr Asp Val Ile Phe Val Thr PheAsn Tyr 115 120 125 cga ttg gga cca ctc gga ttt ttg aat ttg gag ttg gaaggt gct cct 431 Arg Leu Gly Pro Leu Gly Phe Leu Asn Leu Glu Leu Glu GlyAla Pro 130 135 140 gga aat gta gga tta ttg gat cag gta gca gct ttg aaatgg acc aaa 479 Gly Asn Val Gly Leu Leu Asp Gln Val Ala Ala Leu Lys TrpThr Lys 145 150 155 gaa aat att gag aaa ttt ggt gga gat cca gaa aat attaca att ggt 527 Glu Asn Ile Glu Lys Phe Gly Gly Asp Pro Glu Asn Ile ThrIle Gly 160 165 170 175 ggt gtt tct gct ggt gga gca agt gtt cat tat ctttta ttg tca cat 575 Gly Val Ser Ala Gly Gly Ala Ser Val His Tyr Leu LeuLeu Ser His 180 185 190 aca acc act gga ctt tac aaa agg gca att gct caaagt gga agt gct 623 Thr Thr Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln SerGly Ser Ala 195 200 205 tta aat cca tgg gcc ttc caa aga cat cca gta aagcgt agt ctt caa 671 Leu Asn Pro Trp Ala Phe Gln Arg His Pro Val Lys ArgSer Leu Gln 210 215 220 ctt gct gag ata tta ggt cat ccc aca aac aac actcaa gat gct tta 719 Leu Ala Glu Ile Leu Gly His Pro Thr Asn Asn Thr GlnAsp Ala Leu 225 230 235 gaa ttc tta caa aaa gcc cca gta gac agt ctc ctgaaa aaa atg cca 767 Glu Phe Leu Gln Lys Ala Pro Val Asp Ser Leu Leu LysLys Met Pro 240 245 250 255 gct gaa aca gaa ggt gaa ata ata gaa gag ttcgtc ttc gta cca tca 815 Ala Glu Thr Glu Gly Glu Ile Ile Glu Glu Phe ValPhe Val Pro Ser 260 265 270 att gaa aaa gtt ttc cca tcc cac caa cct ttcttg gaa gaa tca cca 863 Ile Glu Lys Val Phe Pro Ser His Gln Pro Phe LeuGlu Glu Ser Pro 275 280 285 ttg gcc aga atg aaa tct gga tcc ttt aac aaagta cct tta tta gtt 911 Leu Ala Arg Met Lys Ser Gly Ser Phe Asn Lys ValPro Leu Leu Val 290 295 300 gga ttc aac agc gca gaa gga ctt ttg tac aaattc ttt atg aaa gaa 959 Gly Phe Asn Ser Ala Glu Gly Leu Leu Tyr Lys PhePhe Met Lys Glu 305 310 315 aaa cca gag atg ctg aac caa gct gaa gca gatttc gaa aga ctc gta 1007 Lys Pro Glu Met Leu Asn Gln Ala Glu Ala Asp PheGlu Arg Leu Val 320 325 330 335 cca gcc gaa ttt gaa tta gcc cat gga tcagaa gaa tcg aaa aaa ctt 1055 Pro Ala Glu Phe Glu Leu Ala His Gly Ser GluGlu Ser Lys Lys Leu 340 345 350 gca gaa aaa atc agg aag ttt tac ttt gacgat aaa ccc gtt cct gaa 1103 Ala Glu Lys Ile Arg Lys Phe Tyr Phe Asp AspLys Pro Val Pro Glu 355 360 365 aat gag cag aaa ttt att gac ttg ata ggagat att tgg ttt act aga 1151 Asn Glu Gln Lys Phe Ile Asp Leu Ile Gly AspIle Trp Phe Thr Arg 370 375 380 ggc att gac aag cat gtc aag ttg tct gtagaa aaa caa gac gag cca 1199 Gly Ile Asp Lys His Val Lys Leu Ser Val GluLys Gln Asp Glu Pro 385 390 395 gta tat tat tat gaa tat tct ttc tct gaaagt cat cct gca aaa gga 1247 Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser Glu SerHis Pro Ala Lys Gly 400 405 410 415 aca ttt ggt gac cat aac ttg act ggagca tgt cat ggt gaa gaa ctt 1295 Thr Phe Gly Asp His Asn Leu Thr Gly AlaCys His Gly Glu Glu Leu 420 425 430 gtg aat tta ttc aaa gtc gag atg atgaag ctg gaa aaa gat aaa ccg 1343 Val Asn Leu Phe Lys Val Glu Met Met LysLeu Glu Lys Asp Lys Pro 435 440 445 aat gtt tta tta aca aaa gat agg gtactt gct atg tgg acg aac ttc 1391 Asn Val Leu Leu Thr Lys Asp Arg Val LeuAla Met Trp Thr Asn Phe 450 455 460 atc aaa aat gga aat cct act cct gaagta act gaa tta ttg cca gtt 1439 Ile Lys Asn Gly Asn Pro Thr Pro Glu ValThr Glu Leu Leu Pro Val 465 470 475 aaa tgg gaa cct gcc aca aaa gac aagttg aat tat ttg aac att gat g 1488 Lys Trp Glu Pro Ala Thr Lys Asp LysLeu Asn Tyr Leu Asn Ile Asp 480 485 490 495

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: (a) a nucleic acid molecule that encodes aprotein selected from the group consisting of SEQ ID NO:68 and proteinsthat are at least 95% identical to SEQ ID NO:68, wherein said proteinexhibits carboxylesterase activity; and (b) an isolated nucleic acidmolecule fully complementary to a nucleic acid molecule of (a).
 2. Thenucleic acid molecule of claim 1, wherein said nucleic acid moleculeencodes a protein comprising an amino acid sequence SEQ ID NO:68.
 3. Thenucleic acid molecule of claim 1, wherein said nucleic acid molecule isselected from the group consisting of: SEQ ID NO:67, SEQ ID NO:69, SEQID NO:70, and SEQ ID NO:71.
 4. A recombinant molecule comprising anucleic acid molecule as set forth in claim 1 operatively linked to atranscription control sequence.
 5. A recombinant virus comprising anucleic acid molecule as set forth in claim
 1. 6. A recombinant cellcomprising a nucleic acid molecule as set forth in claim
 1. 7. A methodto produce a carboxylesterase protein, said method comprising culturinga cell capable of expressing said protein, said protein being encoded bya nucleic acid molecule of claim 1, part (a).
 8. An isolated proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:68 and an amino acid sequence 95% identical to SEQ ID NO:68,wherein said isolated protein has carboxylesterase activity.
 9. Theprotein of claim 8, wherein said protein, when administered to ananimal, elicits an immune response against a carboxylesterase protein.10. The protein of claim 8, wherein said protein is encoded by a nucleicacid molecule selected from the group consisting of: SEQ ID NO:67 andSEQ ID NO:70.
 11. A method to identify a compound capable of inhibitingflea carboxylesterase activity, said method comprising: (a) contactingan isolated flea carboxylesterase protein of claim 8 with a putativeinhibitory compound under conditions in which, in the absence of saidcompound, said protein has carboxylesterase activity; and (b)determining if said putative inhibitory compound inhibits said activity.12. A test kit to identify a compound capable of inhibiting fleacarboxylesterase activity, said test kit comprising an isolated fleacarboxylesterase protein of claim 8 having esterase activity and a meansfor determining the extent of inhibition of said activity in thepresence of a putative inhibitory compound.